text:
Hire Business Lunch Catering in Kegworth
Browse Business Lunch Caterers
Recent review: Debbie & Corinne (sorry if I've spelt that wrong) did a fab job with our work social BBQ. The food was fab, everyone, even the long term burger n hotdog people tried and loved the food - we even had a very happy and full vegan which is pretty unusual for a BBQ. The girls were a great laugh too yet s
Recent review: Great service and lovely food. Adam was fabulous, responded quickly to email queries in the lead up to the event was fine with me messing about with my numbers as my group changed, was on time, pre-prepped and got on with cooking while we finished off our afternoon activities. Tasty BBQ meal for 20
Recent review: I booked Five catering for a joint 40th party for my fiance and his friend. My guests commented on how nice the food was. The caterer was very pleasant and very accommodating. Highly recommended. Thankyou again. ☺
About: About Greens Catering Services Greens are the chosen caterers to the Loughborough Town Hall, Magistrates Courts as well as a number of other blue chip businesses within the Leicestershire area. From sit down meals with silver service, hot or cold buffets served in chaffing dish form, canapes,
About: Juicy Jackets are the UKs premier traditional jacket potato catering specialists. With nearly 10 years experience, Juicy Jackets has become one of the most popular catering concepts around. If you are looking for a stylish and quirky catering choice for your wedding, corporate or private event, then
Recent review: "On behalf of all of us at the Air Ambulance, I would like to thanks you very much indeed for providing the catering of our Silver Service lunch last Thursday, 29th April. The day went exceptionally well, despite the turn in the weather and the slight delay in the Duke's arrival. I wanted to than
About: Street Food providing awesome gourmet toasties ranging from Ultimate American Classic Grilled Cheese, Don't Go Bacon My Heart, Viva Mexico, Italian Stallion, Mediterranean Melange. Menu is adapted to suit the event. We also add Halloumi Fries with a range of toppings.
About: Our family has been in the catering trade for over 25 years. We want to provide a catering service to clients that gives them that attention to detail that a family member would offer. We work with you to ensure that you get the perfect menu for your event. We love a variety of foods and our menu
About: Bespoke pan-Asian premium catering service from chef and best-selling author, Kwoklyn Wan. Available for corporate hire and private parties across the Midlands. We specialise in Asian inspired food from a range of countries; including Chinese, Japanese, Korean and Vietnamese. Our party food and stre
About: Hello from Sky and Emma, the proud owners of Edwin's. We are able to bring a whole lot of Trinidad sunshine and flavour to any event that you are looking to cater from weddings to dinner parties, and from corporate lunches to birthday celebrations. All of our food is 100% vegetarian that can be
About: We are a family business supplying Authentic Indian Food. We are based in Leicester City Centre, we serve to Cafe's, News Shops, Delicatessen shops, many businesses and we take part in many local events and festivals. We make Samosas, Onion Bhajis, Spring Rolls, Veg and Non Veg Curries. Our veget
Recent review: Just to say thank you for a really delicious hog roast last night and truly brilliant service from Ross. He managed everything with such calm efficiency and good humour which was appreciated be everyone. All the food disappeared, apart from a few veggie burgers which I knew I had probably over order
About: At Satya we indulge your desire for exquisite hand crafted food that looks sensational & tastes divine. A special occasion should feel magical. The right Catering is imperative in helping to create a wonderful, powerful, memorable feeling & experience, hence why at Satya we take pride and care in t
About: The Pudding Pantry brings together a small passionate team with an insatiable desire to provide the kind of food and coffee that will keep you coming back. Bringing you the taste of home baking and reminiscing over those days of licking the cake bowl clean, our aim is to create a haven for those w
About: We are an independent business based in Nottinghamshire. We offer fresh, tasty affordable food with a variety of different food. We can cater for all needs.
About: Lunch Angels is a new concept for your catering needs whether for your Office Lunch or a Party, Wedding or Bereavement Funeral wake Buffets in and around Derby, Nottingham & Burton on Trent.
About: We founded Rafiq Catering with the idea of bringing our family's' best recipes to the forefront of the local Leicester real food movement. We specialise in using farm-fresh ingredients and old-fashioned techniques to create home cooked meals that are delicious, healthful and oh so satisfying. As
About: We are a family business based in Leicester, UK and have developed a reputation for quality, exquisite Asian Vegetarian Cuisine.
About: The Wolf Hut serves Wholesome Original Lean Food - specialties are Hot Bagels and Wolf Trays; chilli, curry etc, vegan, veggie and meaty
About: We pride ourselves in making the ultimate Grilled Cheese Toasties,and Gourmet sandwiches. We can serve individually or as a group. We source our products with care, trying to have the best quality possible. We also offer a Bar service.


text:
When you are renting a 5, 10, 15, 20, 30 or 40 yard dumpster, you want a company you can trust with prices that make you smile. Give us a call today and see the difference we can make in your next construction or clean out project.
Simply give us a call and we will help you figure out your dumpster rental needs.
Our dumpsters usually go out same-day or next-day depending on when you call.
We provide top-notch service, while going easy on your bottom line. What more could you ask for?
Our trained operators are here to give you a fast and hassle-free experience from start to finish.
Once you are done, simply give us a call and we will get your dumpster picked up right away!
Our staff is here to help whenever you need it. Simply pick up the phone and give us a call.
Most cities or municipalities do not have many regulations regarding dumpster rental as long as you keep the dumpster entirely on your own property during the rental period. If you need to place your container on the street in any way, you will likely have to obtain a permit from the appropriate building permit office in your town. You can check with your local public works department to be sure.
Most Port Hope dumpster rental companies will take care of securing this permit for you if you rent from them. Make sure that if you're planning to put the dumpster on the street, the company has made the appropriate arrangements. You should also ensure that you get the permit in a timely manner and at the correct cost. If you thought the dumpster company was getting a permit and they did not, you will be the one who will have to pay the fine that is issued by the authorities.
One of the most important facts you must consider in terms of Large Dumpster For Rent in Port Hope, MIs is that size is essential. You'll find that dumpsters come in many different styles and you'll desire to choose the suitable size for your requirements. While you must ensure that it is big enough to hold all the trash you should have until pick up, you don't want to buy something that is just too big large for your requirements either. Evaluate the dimensions of the particular dumpster before choosing. Likewise, consider the distinctions between top load along with roll off of containers an internet to choose the suitable rental for your spend management needs.
Compulsive holding on to is seen as the inability as well as unwillingness to remove trash from your home, even if the items becoming kept have no value, tend to be unsanitary, as well as actually hinder on the client's ability to execute everyday jobs. Social interactions, professional ambitions, mobility in your home, and even day-to-day necessities such as cleaning and cooking can be made hard or impossible by the abnormal amounts of hoarded crap. Beating this issue requires healthcare intervention along with counseling, in addition to moral assistance from close relatives and other loved ones. Before those steps is usually fully realized, however, you have to first make sure that the living area in question is completely removed of all unneeded garbage, specifically any things that may be specifically threatening private safety as well as posing a serious risk for the property. In order to take this vital action, hoarders may require the assistance of a professional crap hauling organization.
The weather outlook may be with rain as well as snow and you need to get the cover job done next few days. Thank goodness you can get your own supplies speedily such as the roof shingles for the roofs and a dumpster the very next day.
You need to know the type of services some sort of dumpster company delivers before renting some sort of dumpster. Many dumpster companies perform roll-off-services, the good way of getting loads of function done without fretting about where the junk will go. A firm using roll-off providers will take out the dumpsters along with replace them with new ones anytime it will be required. In addition, a lot of companies use the huge dump pickup trucks to remove the spend collected in the dumpsters leaving vacant dumpsters to be used. In case you a business owner and you have a lot of spend, you will definitely want to be in agreement that has a Large Dumpster For Rent in Port Hope, MI company. By doing this, you will end up protecting your business a ton of money.
Your living area is your getaway and you may complete a lot of be employed in there. Once in awhile you may want room for any comfortable extra-large leather couch. If you decide to redesign your living area, than you can do this so that you are satisfied with your living area.
How long does one get to keep dumpster? Most companies will let you keep your dumpster for between 5-7 times. If you're lucky, you might find an organization that will let you keep your dumpster for as much as 2 weeks. What are the results if you want to ensure that it stays longer than your own agreed timeframe? Extra fees. Generally, you will incur a daily charge charge everywhere you go from $5 in order to $15 per day, as soon as the initial rental period of time.
Many house projects are generally started without planning for correct debris disposal. Usually, mass waste is actually carted out to the actual curb hoping that one day the city will eliminate these items and dispose of these individuals at the community landfill or even disposal service. While every single municipality is different, many currently have specific regulations regarding the disposal of large house or design debris. Once you understand the regulations, it may be some time before the merchandise is actually acquired. They will reside on the curtail in front of your property, attracting vermin and appearing a safety threat to local kids until the city clean up group can prepare to pick all this up.
It doesn't matter what project you have in your home to finish, you should rent the dumpster to complete the job in a organized and quick way.
People often find themselves in generating large tons of garbage on finishing their projects. Piling up garbage for longer time frame not only punches you in worries but also makes you a source of pollution generation. You've got to take a sudden action to wash off the garbage and recycling it an eco friendly approach. How will you make this happen? A dumpster use from a professional rental service provider will make your task of recycling the trash pretty straightforward.
Have you considered your garage area or underground room lately? Almost all of us use these locations as everlasting home hard drive for points we think organic beef eventually make use of. But there will always be more points we could maybe need in the foreseeable future, and these spots fill up easily, often to where we weren't able to find any of the things we need between the clutter.
When appliances can be parted available and the recyclable metals segregated for use or even sale, then a old package can be cast into the dumpster and this will get acquired when you're performed. On the other hand, when appliances will be in decent appearance then you can spot those, in addition any other reusable whole cabinetry or other items in a small dumpster and donated these individuals to a non-profit, like Habitat Pertaining to Humanity's resale shop. Online Large Dumpster For Rent in Port Hope web-sites are the best useful resource known to man and woman, at least, should they be in the design business.
Victorian houses are simply stunning especially when these are kept up. Whenever remodeling nearly all Victorian homes you have to follow the guidelines and get a new one with the same product or service as yesteryear. Keeping the house restored to the original manner is usually the concept in most areas.
The second reason the Large Dumpster For Rent in Port Hope may be required is the number of waste you may be producing, roofer an average house can produce way up to 9 tons of waste alone. Sheetrock, old household furniture, and roof shingles also weigh more in the collective perception than nearly all would feel. This tonnage may typically exceed the limits of normal assortment. Dumpster rentals are cheaper than ever before and can be delivered to the position site and picked up at the same time. These components alone allow it to be mandatory for almost all project administrators to incorporate waste management in their plans at the beginning. Forgetting to accomplish that is a head ache many home-owners do not wish to currently have. Dumpster rentals make liability and hassle far from dealing with the various permits and legalities necessary with big disposals. Furthermore, having dumpsters today being so effortlessly delivered and removed, that is likely the most cost effective way to eliminate items out of your to do number. Most dumpster locations even provide to help eliminate items with the work site for an extra fee. Determined by what type of waste is being put together by the challenge some firms may even provide to take it free of charge. This is very beneficial and implies that one person's trash is perhaps another's prize. Many design projects don't make use of these facilities and get knotted up in red tape as it is. The benefits of using a community Large Dumpster For Rent in Port Hope company are generally significant.
Early in the year you may want to begin school remodeling that need to take place now. In the future, schools are becoming more and more crowded. Additions are necessary as well as school room sizes made larger. Through spring break you can have a contractor arranged to start the actual renovations. To acquire the remodeling done quickly you will need to hire a company that the cost of rent next day dumpsters. Your contractors can readily do their work and also clean up soon after with a dumpster right next to these individuals. Make sure you cope with a company containing dumpsters with wheels on the bottom. Your contractors should be able to easily move the dumpster from one method to another while working.
Coping with these big tons of trash on self would be a headache and requires great deal of investment in relation to time and money. Your current painstaking research to find an inexpensive way of getting rid of the trash inside the shortest moment possible would likely end up in Large Dumpster For Rent in Port Hope, MI service.
Dumpsters are usually accessible in different the actual sizes that will range between 10 garden and Forty yard. You should be extra careful while seeking the size of dumpster due to the fact choosing the incorrect sized dumpster other than required will impose extra charges as you have to go for several pickups to help dispose your current trash should the dumpster is smaller in size. You're going to be paying for the disposable space when you purchase a dumpster that's large in space.
A major cleanout is usually stressful and also exhausting. Whether or not you're planning to have an upcoming cross-country move into a smaller household, cleaning out an older relative's home ahead of an real estate sale or perhaps foreclosure, or perhaps gut-renovating your office and also installing all-new accessories, all of the trash you pitch has to head out somewhere. You can divide the garbage and trash up directly into small lots and get back and forth on the dump as well as the recycling heart, wasting time, fuel, as well as. Or you can save the hassle by simply hiring a expert junk carrying team to accomplish the hard work and convenience for you. Precisely what does a trash hauler do? It all depends on the specific company, and so consider your requirements and your budget before making a using the services of decision.
Whenever you embark on a major cleaning, development, or redevelopment project, the final thing you want to be worried about is getting lessen the causing debris. Doing the work is hard sufficient. There's no answer why you should be burdened with baggage and loads of trash afterward. These could clutter the property, which will directly negates the purpose of doing the work to start with and can require special journeys to the remove or other convenience facility afterwards. Instead of fixing your trash removal in the piecemeal way, it might be worth your while to review renting a dumpster. While wearing a dumpster in your yard may seem like a step backwards, it really simplifies the actual cleanup procedure. You can rent a dumpster for as long as you would like, you can put all your trash and also debris inside, and then you could possibly get rid of all things in one chop down swoop if you are done. In case you are doing a residence clean-out, construction washing, or other do-it-yourself or redevelopment projects, renting a dumpster can be a good option.
Please use the form below to contact us. We look forward to working with you and will get back to you as soon as possible. All fields are required

subset: commoncrawl


text:
Used for use in schools and gym locker rooms. These premium metal storage lockers feature 16 gauge steel doors, 5-knuckle security hinges and welded, reinforced frame-and-door assemblies for maximum security. School lockers feature extra wide louvers to promote ventilation. Legs add 6" to height. Durable, attractive powder coat finish is available in the 3 most popular school locker colors: blue, tan and gray. These gym lockers include blank number plates. These school lockers feature finished sides; no need for decorative end panels. Optional locks, numbered plate kits and bases sold separately.
Lyon storage lockers have a padlockable hasp as an integral part of the handle. Legs add 6 to height. Single and Double Tier Steel Lockers accept either padlocks or built-in locks (sold separately). Single Tier Steel Lockers are the most practical and widely used of all storage lockers. 12 D & 15 D School Lockers have a center ceiling double prong hook. 18 D & 21 D Single Tier Lockers include coat rods for hanging clothing. Double and Triple Tier Office Lockers are great for short-term use and storage of shorter length garments. These metal lockers include one double-prong and three single-prong coat hooks. Four, Five, or Six Tier Lockers are used for the storage of high density or individual belongings and supplies. Lyon recommends anchoring steel lockers to floor and/or wall for safety. Optional Number Plates can be added to all lockers for quick identification. Assembled storage lockers are non-returnable. Lyon storage lockers are available in Gray or Putty. Limited Warranty.
Lockers Aurora Colorado 80015
Shelving: We have 3 types of shelving available. Our sheving is popular in business, factory, and industrial settings as well as great for storage in and around the home and garage. Our shelving units are especially popular in restaurants and medical settings. Solid shelving, stationary wire shelving, and mobile wire shelving are available with chrome or black finishes (wire shelving) and all shelving units are delivered unassembled for quick shipment and low delivery costs. Denver Lockers
Fear mongering and lying about what is meant by Democratic socialism is the only way the party of greed and racism can win IMO. The Dems are failing by not repeatedly clarifying what is meant by Democratic socialism, which already exists in many areas and is fully compatible with capitalism, just not with a corporate-run government, but by a gov't of, by, and for the people.Other GOP tricks include lying about the Democrats' plan for fully open borders, letting anyone in without screenings, etc. And then there are women's rights. Along with bigotry, here's an example of a Christian college's censorship of art, using books where all offensive body parts are covered -- this would be funny if it weren't so sick! https://www.distractify.com/p/censored-art Lockers Denver Colorado
To see if ShippingPass is right for you, try a 30-day free trial. Also, with ShippingPass, there is no need to worry about commitment. If you decide you want to discontinue the service, you can cancel your subscription at any time. No matter what your shipping needs, Walmart's got you covered. Sign up for ShippingPass so you can shop more, save money and live better.
Lockers Denver 80239
Penco school lockers feature a stainless steel recessed handle that provides a smooth, catch-free front and protects padlocks from being tampered with. Locker doors feature spot welding for rigidity. Doors are louvered for ventilation. Office storage lockers include continuous door strikes to ensure secure closure and rubber padding to dampen closing noise. Metal lockers have 5-knuckle hinges for strength. Latching channels have nylon glides to reduce sound when opening or closing. Legs add 6" to height. Single Tier Office Lockers have 3-point latching (top, bottom and sides). 18 D lockers include coat hooks and a coat rod. Double and Triple Tier Employee Lockers have 2-point latching (top and bottom). Includes coat hook. It's recommended to anchor steel lockers to floor and/or wall for safety. Powder coat finish. Denver Lockers
Heavy Duty Ventilated Lockers are designed to meet the harsh requirements of an athletic room environment. Built to last, these lockers feature rugged, 14 gauge steel doors, a heavy gauge body and a premium paint finish throughout. The Heavy Duty Ventillated Lockers offer clean, contemporary appearance, reliable security and rugged, heavy duty construction for years […]
Lockers Den Co 80237
ASI Storage Solutions® Traditional Single Tier Ready To Assemble Lockers have an extra heavy duty 2mm powder-coated finish to prevent corrosion. 16-gauge steel door, frames and bottoms and 24-gauge steel tops, sides, backs and shelves provide long-lasting durability. Features stainless steel recessed handle for safety. Rubber door silencers for quiet operation. Continuous full-length piano hinge and multi-point latch for ease of use and security. Legs add 6 inches to height. Available in Almond, Blue Frost, Burgundy, Gray, Mist Green and Black. Dimensions per model. One Year Limited Warranty.
Lockers Denver Co 80290
Provides long article height in less floor space yet offers full width private compartment for each user. Locking device in coat compartment opens full width hat/book compartment above. Equipment: Coat hooks (rod in 19″ depth), number plates and padlock attachment. Widths Depths 12″ 12″ 15″ 18″ 21″ 18″ 15″ 18″ Opening Heights available: 20″, 24″. […]
Don't see exactly the lockers you are looking for on our store? Please contact us at 1-877-909-9910 and our customer service team can give you suggestions and point you in the right direction on our lockers for sale, so that you order the correct product the first time around. Also check out our other AV equipment such as our speakers and PA Systems that can add to your presentations.
Lockers Denver 80211
Single point lockers are designed for facilities where users are especially tough on lockers. A reinforced, double panel door resists prying and tampering and promotes quiet, reliable operation. When a padlock is used for locking, the Single Point Locker is virtually maintenance free. Engineered and built to last, Republic's Single Point Lockers provide the security […]
Lockers Den Co 80202
Lyon Ready To Assemble Steel Lockers are solidly constructed for long lasting durability. Lyon storage lockers include a padlockable hasp (lock sold separately) within the recessed handle to provide a catch-free surface. Single Tier Steel Lockers are practical and widely used as school lockers. Two Person Lockers feature 2 compartments for each occupant. Four, Five and Six Tier Box Lockers are used for the storage of high density or individual belongings and supplies. Lyon recommends anchoring steel lockers to floor and/or wall for safety. Single or double prong coat hooks available with some models. Available types are One to Six Tier, Four Person Wall, Two Person, and 16 Person Lockers. Color finishes are Gray and Putty. Includes Lifetime Limited Warranty. Dimensions vary per model.
Lockers For the Home: Our metal lockers help to keep things tidy in your home, closet and garage. Our modular lockers are popular in the home and great for organizing. We have many residential customers. There are many applications of these units as they can be combined and arranged in various ways. Our storage cabinets make a great addition to the garage and really help organize and keep the dust off items.
Any school that has sports teams, especially those that require bulky equipment, should consider investing in athletic lockers. Hockey and football players need a lot of large padding in order to stay properly protected, and it is important to make sure that they have a good place to store it so they won't forget anything. We have basic athletic lockers as well as enhanced models with lockboxes and footlockers. Denver Lockers
Quality Refurbishing Many of the old, wholesale lockers we have in stock have minimal damage and some are near brand-new condition. For lockers that need repairs, our unique multiple point inspection and repair process helps to refine these lockers to a high quality, salvaged state. We know you want to buy quality lockers and we will not be shipping out lockers that are not fully functional. To get the most out of your refurbished locker, we also offer tips on alternative ways you can utilize them. Don't want to have the same old wall locker look? Use one as a laundry room locker or a console table.
In the States everybody is so concerned about liability.... We tried to store at Hertz (where we dropped the rental); no storage facility, no luck. Try a hotel; I tried several, if you are not a guest then they are not allowed. I tried the Information Centre; no luck, they had some bad experiences.... in the past. But they could not realy help me any further. One advise: try the Greyhound Bus Center. And yes! there you can store your luggage. Even my somewhat bigger suitcase. The lockers are somewhat small but high and deep enough. First 3 hours is to be paid for; $ 3 and after that a dollar for an extra hour. Denver Lockers
ASI Storage Solutions® Traditional Triple Tier And Above Ready To Assemble Lockers have an extra heavy duty 2mm powder-coated finish to prevent corrosion. 16-gauge steel doors, frames and bottoms and 24-gauge steel tops, sides, backs and shelves provide long-lasting durability. Features stainless steel recessed handle for safety. Rubber door silencers for quiet operation. Continuous full-length piano hinge and multi-point latch for ease of use and security. Legs add 6 inches to height. Available in Almond, Blue Frost, Burgundy, Gray, Mist Green and Black. Dimensions per model. One Year Limited Warranty.
Lockers Denver Colorado 80204
Heavy Duty Ventilated Lockers are designed to meet the harsh requirements of an athletic room environment. Built to last, these lockers feature rugged, 14 gauge steel doors, a heavy gauge body and a premium paint finish throughout. The Heavy Duty Ventillated Lockers offer clean, contemporary appearance, reliable security and rugged, heavy duty construction for years […]
Lockers Denver Co 80219
We offer heavy duty, sturdy lockers in a variety of sizes and tiers for your convenience, as well as lockable lockers so that your items are safe and secure even when you are not around. These lockers fit well in offices, vocational learning rooms, gyms or even preschools. We offer metal lockers from leading manufacturers Sandusky Lee, Shain and Diversified.
Lyon has long been America's leading manufacturer of quality lockers for schools, hospitals, and industry. Wherever you go – either on campus or on the shop floor, in police, fire and municipal buildings, on military bases, in hospitals and nursing homes, or the back rooms of retail stores. Besides standard metal lockers we have other great products. From sports lockers and gym lockers to gear or employee lockers. Contact us today to speak with an expert. We can help you come up with a custom locker solution for you project.
We have metal lockers for sale, yes, metal lockers that are designed for many applications like first responders of all types, school corridor lockers, employee lockers, high school lockers, Athletic lockers Footlockers, and Stadium® lockers that are ideally suited for athletic team rooms to store helmets, uniforms and gear for many different sports.
Digilock LockUp electronic access door employee steel lockers are made of steel for long lasting durability. Lockers provide visible and secure storage with user friendly 4 digit code electronic entry locks. Doors are mounted to locker frames with continuous hinges for unit stability. Includes one Manager Flex Key allowing management locker access or ability to power jump a lock when battery is no longer operational. Available types include Six Tier and Triple Tier. Groups are 1 Wide and 3 Wide. Door types are Louvered and Polycarbonate. Color finish is Gray. Dimensions vary per model. Lockers Denver Colorado
There are no lockers, at present. If you are taking the Amtrak, you can, of course store your bags with them. If you are staying at the Crawford or he Oxford, or the Indigo, you should leave your bags with them. If you are just waiting for theA line to DIA or the Light rail, you'll have to watch you own bags, but the Main Reception Hall has plenty of couches and chairs and food and rdink and chotckes. It is good for hanging out and playing shuffleboard.
Lockers Den Co 80239
Assembled storage lockers include one combination lock per door for maximum security; eliminates need for users to purchase their own locks. 3-number combination locks are master keyed for back-up emergency entry. Steel lockers are made using 24 gauge steel body and 16 gauge steel locker doors with stainless steel recessed handles; Six Tier Lockers feature 18 gauge doors with padlockable hasps and aluminum strike plates that prevent padlocks from marring finish. Doors are louvered for ventilation and secured using continuous piano hinges. Sloped top provides a finished appearance that prevents clutter on top of lockers. Closed base encloses locker to prevent debris collection under locker. Single Tier Lockers include a top shelf for added storage. Single & Double Tier Lockers include one double and two single prong coat hooks. Triple Tier Lockers include a double prong hook. Choose between Gray, Putty or Blue finish. 2 Year Limited Warranty.
We offer lockers of all types and sizes for your school, office, gym or preschool. Choose from top brands like Lyon Metal and Hallowell both with a long history in the locker and storage industry. Create organized storage for individuals in places such as gym locker room, warehouse, police departments, hospitals or any facility that requires heavy-duty personal storage. Many make great coat storage as they feature hooks and shelves for coats and other garments. Available in several styles and materials, the school lockers are constructed of metal and are specifically designed for daily use in school hallways. Wood lockers are often used in private gyms, sport clubs or college athletic departments as they have a more custom built look. Lockers Denver Colorado


text:
There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.
Citation: Gunnarsen KS, Høydahl LS, Neumann RS, Bjerregaard-Andersen K, Nilssen NR, Sollid LM, et al. (2018) Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system. PLoS ONE 13(4): e0195868. https://doi.org/10.1371/journal.pone.0195868
Editor: Marek Cebecauer, J. Heyrovsky Institute of Physical Chemistry, CZECH REPUBLIC
Received: January 24, 2018; Accepted: March 30, 2018; Published: April 12, 2018
Copyright: © 2018 Gunnarsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The study was funded with grants from South-Eastern Norway Regional Health Authority (grant 2012046, https://www.helse-sorost.no/, KSG), the Research Council of Norway (grant number 197085, https://www.forskningsradet.no/en/Home_page/1177315753906, LSH), and the Research Council of Norway through its Centers of Excellence funding scheme, project number 179573/V40, LSH and GÅL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. GÅL is in part employed by the commercial company Nextera AS. The funder provided support in the form of salary for author GÅL, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the 'author contributions' section.
Competing interests: GÅL is in part employed by the commercial company Nextera AS that works with T cell receptor engineering. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
The T-cell receptor (TCR) plays a central role in adaptive immunity by mediating recognition of peptides presented by the major histocompatibility complex (MHC) on the surface of antigen presenting cells. Studies of the interaction between individual TCRs and their specific peptide MHC (pMHC) complexes continue to give insights into the biological functions of T cells, as well as information necessary for the design and safeguarding of TCR-based therapeutics [1–3]. Production of soluble TCRs of high quality and in high yields is therefore needed for biophysical characterization of TCR interactions. Furthermore, soluble TCRs are useful as detection reagents when studying antigen presentation [4–6]. Compared to the structurally similar antibody molecules, TCRs are generally less stable when expressed as soluble molecules, and problems such as low expression yields, aggregation, misfolding and inefficient chain pairing are often encountered [7–9].
Different soluble TCR formats have been constructed, such as single-chain TCR (scTCR), where the two variable domains are connected through a flexible linker, fusion of the TCR α- and β-chains to other proteins, such as leucine zippers, the human constant antibody kappa domain or the TCR constant β domain, and by introduction of a non-native disulfide bond between the TCR constant domains to generate disulfide-linked TCRs (dsTCRs) [10–15]. Production of dsTCRs has been the most successful strategy, but the molecules have only been expressed in the E. coli cytosol, and thus at reducing conditions not compatible with efficient disulfide bond formation and chain pairing. An attempt to overcome this by use of a modified E. coli strain had limited success, and one therefore relies on refolding of cytosolic inclusion bodies for production, which is time-consuming and not compatible with high-throughput approaches [13, 16].
High throughput approaches are desirable whenever engineering is carried out to increase the stability, affinity or production yields by phage or yeast display. Therefore, an attractive alternative is protein targeting to the oxidizing periplasm of E. coli with simultaneous co-expression of chaperones such as FkpA, and which is compatible with large-scale library screening after display selections [17]. Such periplasmic targeting has resulted in greatly increased functional expression yields of several scTCRs [17–20]. However, whether or not other TCR formats can be produced by periplasmic targeting, has not been investigated.
In the current study, we wanted to study how different TCR formats were expressed after targeting to the periplasmic space with FkpA folding assistance. We aimed to produce high quality, soluble TCR for biophysical studies of the TCR-pMHC interaction. To this end, we constructed soluble TCRs of 4 different formats, namely the two previously described formats, scTCR and dsTCR, as well as two novel formats, which we denote cFab (chimeric Fab) and ct-dsTCR (c-terminal dsTCR). We also included a scTCR variant containing stabilizing mutations. We compared the expression yield, stability and purity of the variants, as wells as the importance of expression host (XL1-Blue or RosettaBlueTM) and expression conditions with or without IPTG induction. We observed expression of all formats and variants, and identified the most successful combination of format and conditions. The stabilized scTCR expressed in RosettaBlueTM without IPTG induction, enabled production of sufficient quantities of soluble TCR for pMHC interaction studies.
TCR construct design
In order to obtain soluble monomeric TCR of high quality and yield using expression conditions that are compatible with high throughput production of variants, we compared periplasmic E. coli expression of different molecular formats with co-expression of the chaperone FkpA [17, 20]. Using the celiac disease-associated HLA-DQ2.5:DQ2.5-glia-α1a-restricted TCR380 as template [21], we constructed 5 variants as schematically illustrated in Fig 1A. The scTCR construct was generated by connecting the variable domains of the TCR with a flexible linker in a Vα-linker-Vβ orientation which was found to be the preferred orientation in a previous study [20]. We generated two variants of this format, wt380 and a stabilized version, s380, containing mutations predicted or shown to impact on stability (Fig 1A and detailed in S1A and S1B Fig) [17, 22]. The dsTCR was generated by introducing a disulfide bridge in the invariant region of the constant domains of the TCR [13], while the ct-dsTCR was engineered to contain the disulfide bridge-containing region from the antibody Fab fragment (Fig 1A and detailed in S1C and S1D Fig). Finally, the cFab format, a TCR/antibody chimeric Fab (cFab) fragment, was constructed by fusing Vα from the TCR to CH1 and the Vβ to CK (Fig 1A and detailed in S1E Fig) [23, 24].
(A) Schematic presentation of the TCR formats. From left to right; scTCR wt, scTCR s, dsTCR, ct-dsTCR and cFab. TCR α- and β-chains are colored pale green and purple, respectively, and CH1 and Cκ of the cFab are colored red and blue, respectively. The Vα/Vβ domains of the scTCRs are connected via a peptide linker as shown in green. Disulfide bonds are illustrated in yellow and the c-Myc and His6 -tags are shown in grey. (B) Top-down view of the amino acids forming the Vα/Vβ interphase triad, P50TRAV, L50TRBV and F103TRBV. The crystal structure of the HLA-DQ2.5-DQ2.5-glia-α2-specific TCR S16 was used for modelling the triad (PDB ID 4OZH) [25].
Influence of host strain and expression condition on soluble TCR expression
We performed an initial small-scale E. coli periplasmic expression of the 5 different variants. Two different E. coli host strains were compared, XL1-Blue and RosettaBlueTM. XL1-Blue allows for efficient protein production and is a phage display compatible strain [20, 26–28]. RosettaBlueTM is suited for protein production; in particular expression of eukaryotic proteins due to the expression of tRNAs for codons rarely used in E. coli. The variable region genes of TCR380 studied herein contain 16 such rare codons. Furthermore, the effect of including or omitting IPTG induction of protein expression was assessed. Overall, RosettaBlueTM expression without IPTG induction resulted in the highest periplasmic expression yields (Fig 2A, S2 and S3 Figs). Under these expression conditions, heterodimer formation of dsTCR, cFab, and ct-dsTCR was detected. However, the ct-dsTCR band was very weak (Fig 2A and S3 Fig), and in some cases not even visible on the gel (S2 Fig). Thus, the ct-dsTCR was excluded from further analysis due to low yield of heterodimeric protein.
(A) Representative western blots showing the expression profile of the TCR formats in either E. coli XL1-Blue (X) or RosettaBlueTM (R) where IPTG induction of protein expression either was omitted (-) or included (+). Normalized expression cultures were fractionated before the presence of TCR in the periplasmic fractions was analyzed by western blot detection using an anti-His-HRP antibody (n = 3). (B) The bacterial growth characteristics were monitored by measuring the OD600nm of E. coli XL1-blue (X) and RosettaBlueTM (R) bacterial cultures after overnight protein expression without (-) or with (+) IPTG induction at 30°C. n = 3 for all constructs except for controls (n = 4) and the dsTCR constructs (n = 1). Standard deviations are indicated where possible. (A, B) Cells expressing scFv anti-phOx were used as positive control and untransformed cells as negative control.
In general, IPTG induction reduced the expression levels, and for cFab, dsTCR and ct-dsTCR the expression was completely abrogated (Fig 2A). Expression in XL1-Blue was lower than in RosettaBlueTM, both in the presence and absence of induction (Fig 2A). Moreover, IPTG induction resulted in growth retardation in both XL1-Blue and RosettaBlueTM (Fig 2B). Thus, after initial characterization of expression characteristics, both scTCR variants were readily expressed, while formation of the heterodimeric TCR formats resulted in less detectable protein.
Effect of TCR format on expression characteristics
To compare the 4 remaining variants with respect to production yield and purity, we purified His-tagged molecules by IMAC, followed by size exclusion chromatography (SEC). The final monomeric yields varied extensively, giving the hierarchy scTCR s380 (1.15 mg/L), scTCR wt380 (0.76 mg/L), cFab 380, (0.35 mg/L), and dsTCR 380 (0.14 mg/L) (Fig 3A and Table 1).
(A) Representative Superdex 200 SEC profiles of IMAC purified scTCR wt380, scTCR s380, cFab 380 and dsTCR 380 as indicated (n = 2–9). Elution volumes corresponding to the expected size of monomeric scTCRs and heterodimeric cFab 380 and dsTCR 380 are indicated by arrows. Notably, for the dsTCR we pooled several fractions as indicated by the dotted lines due to the lack of a clear peak indicating the elution volume of the dsTCR. (B) Representative non-reducing and reducing SDS-PAGE of SEC purified monomeric TCR fractions (n = 2). (C) Non-reducing and reducing SDS-PAGE of Resource Q/Superdex 75 purified monomeric and heterodimeric TCR fractions (n = 2). (B, C) Bands at about 32 kDa for the scTCRs, 55 kDa and 26/29 kDa for the cFab and 57 kDa and 25/32 kDa the dsTCR depending on non-reducing or reducing conditions are indicated with arrows (blue arrows for heterodimeric cFab and dsTCR, green arrows for scTCR and reduced β-chains and grey arrow for reduced α-chains.
We then analyzed the samples on SDS-PAGE (Fig 3B). Both wt and in particular s380 scTCR contained mostly monomeric protein of expected size. For the cFab, the dominant fraction was the covalent heterodimer. In contrast, the dsTCR was dominated by single chains and only a very weak band of such heterodimeric protein was detected, excluding this format from further analysis.
The remaining 3 variants were subjected to a final purification step by either ion exchange, or a new SEC resulting in highly pure samples. This came at the cost of loss of protein from all samples and in particular the cFab (Fig 3C and Table 1). Thus, after expression and purification the dsTCR and cFab variants were excluded.
Effect of TCR format on protein stability
To investigate aggregation propensity and melting temperatures of scTCR wt380 and s380, we analyzed the isolated monomeric fractions by analytical SEC and differential scanning fluorimetry (DSF) after a freeze-thaw cycle. Despite application of equal amounts of protein of normalized concentration in the analytical SEC, the scTCR wt380 variant showed a reduced area under the curve (AUC) value compared to s380, indicating that a fraction of wt380 either precipitated in the column filter or nonspecifically adsorbed to or interacted with the matrix (Fig 4A). Thus, analytical SEC revealed that scTCR wt380, but not s380, showed signs of aggregation translating to a reduced amount of monomer. The melting curves of the scTCRs were analyzed by DSF and revealed average Tm values of the scTCR wt380 and s380 of 48.7°C and 43.3°C, respectively (Fig 4B and Table 2). Despite the fact that scTCR wt380 has the highest Tm value, the high initial fluorescence indicates that the wt380, and not s380, contained a fraction of partially unfolded proteins (Fig 4B).
(A) Analytical SEC of scTCR wt380 and scTCR s380 to assess the integrity of the preparations after a freeze-thaw cycle (n = 2). Details of aggregates and monomeric peaks are shown in the magnified inset chromatograms. (B) DSF or (C) fluorescence spectroscopy measurements of scTCRs wt380 and s380 to calculate the melting temperature (Tm) of the variants. The Tm values are summarized in Table 2 (n = 9–10 for DSF measurements and n = 2 for fluorescence spectroscopy measurements).
To validate the DSF data, we determined the melting curves of the proteins based on intrinsic tryptophan fluorescence. The spectra revealed a single transition with a Tm of 43.2°C and 39.2°C for the scTCR wt380 and s380, respectively (Fig 4C and Table 2). Thus, both methods show the mutant form to be less thermostable than the wt with a 4°C lower Tm. However, all data taken together points to aggregation of partially unfolded scTCR wt380, while the scTCR s380 remains in a uniform monomeric state.
Functional binding and affinity measurements of the soluble TCRs
Next, we performed interaction studies using surface plasmon resonance (SPR) to determine binding affinity. Both scTCR wt380 and s380 bound their ligand HLA-DQ2.5:DQ2.5-glia-α1a, with apparent affinities of 30 μM and 7.5 μM, respectively (Fig 5A and 5B and Table 3) [21]. Thus, the affinity is in the 1–100 μM range typically observed for gluten-pMHC complexes [25, 29, 30], as well as for microbial-derived peptides [31, 32].
(A) Biotinylated pMHC was captured on neutravidin-coated sensor chips followed by injection of a 2-fold dilution series from 40 μM of scTCR. Representative sensograms of scTCR wt380 and scTCR s380 binding to HLA-DQ2.5:DQ2.5-glia-a1a (n = 1–3). (B) The equilibrium response was plotted against concentration to derive the equilibrium dissociation constant (KD) of the scTCRs wt380 and s380. Dotted line indicates the KD based on fitting the response to saturation. The KD values are summarized in Table 3. (C) Biotinylated HLA-DQ2.5:CLIP2 was captured on neutravidin-coated sensor chips followed by injection of 5 μM or 20 μM of scTCRs. Representative sensograms of scTCR wt380 and scTCR s380 are shown (n = 1–3).
The binding response was higher for scTCR s380 than for wt380 at equal concentrations, indicating that wt380 contained a non-binding fraction. This is in line with the observations from the analytical SEC and DSF, further highlighting the necessity of stability engineering in order to obtain high quality SPR data. Neither of the scTCR preparations bound HLA-DQ2.5 with irrelevant peptide, CLIP2 (Fig 5C).
Discussion and conclusions
Soluble TCRs are generally unstable and difficult to produce as functional molecules. In previous studies we have reported that over-expression of the chaperone FkpA has a major effect on soluble periplasmic expression of scTCRs, display levels on phage as well as selection performance of phage libraries [17, 20, 26]. In the current study, we utilize the same FkpA-assisted expression system and perform a side-by-side comparison of 4 different TCR formats; cFab, dsTCR, ct-dsTCR as well as wt and a stabilized version of scTCR. Furthermore, we investigate the influence of various E. coli host strains and induction of recombinant protein expression by IPTG.
Several E. coli strains are available for recombinant protein production, some of which are engineered for favorable traits. Here, we compared E. coli XL1-Blue and RosettaBlueTM. The latter strain expresses the pRARE plasmid that encodes tRNAs for codons that are rarely used by E. coli, thereby potentially enhancing the translation of eukaryotic genes containing such codons. Furthermore, culture conditions, such as induction, have been shown to influence protein production yields and subcellular localization. Enhanced expression due to IPTG induction of Lac promotor (LacPO)-regulated expression systems has previously been observed to result in decreased periplasmic protein yield [33], presumably due to clogging of the translocation system and overloading of the chaperones [34]. This may explain our observation that IPTG induction led to growth retardation in both XL1-Blue and RosettaBlueTM. In the absence of glucose and IPTG in the growth medium, leaky basal transcription occurs through the LacPO, resulting in reduced host stress due to lower translation and folding load. We have previously shown that these expression conditions are beneficial for soluble periplasmic scTCR expression [20], and here we extend the finding to include dsTCR, ct-dsTCR and cFab, none of which have previously been produced in a soluble periplasmic E. coli expression system. The combined effects of FkpA over-expression and reduced transcription, translation and folding load were essential for successful expression. Furthermore, the expression host RosettaBlueTM generally gave a higher soluble yield than XL1-Blue. Thus, codon optimization or expression in a host engineered for eukaryotic protein expression is advised.
The TCR formats included in this study have different strengths and weaknesses as regards to downstream applications. Chimeric antibody variants, such as the cFab, can be highly useful as targeting unit e.g., as flow cytometric detection reagent in combination with commercially available secondary antibodies. This format, as well as the dsTCR variants, has the potential advantage of high stability, as the two chains are connected through a disulfide bond. However, as formation of disulfide bonds can only be efficiently achieved in the oxidizing environment of the E. coli periplasm, with the exception of using engineered E. coli strains [16], this is also an expression bottleneck. Furthermore, the two chains are expressed as separate polypeptide chains, which can result in differential expression and subsequent inefficient or erroneous chain pairing [35, 36]. Both the cFab and the dsTCRs were expressed as heterodimers. However, a substantial fraction was also detected as unpaired chains or aggregates. Thus, the folding machinery seems to be somewhat overloaded. It should be noted that the cFab format gave a higher yield of heterodimer than the dsTCR variants. The antibody molecule has evolved to be expressed as a soluble molecule, and fusion of unstable proteins to antibody domains has also previously enhances soluble expression, including of TCRs [14, 37]. Indeed, E. coli is capable of folding even the complex full-length antibodies in the periplasm at levels comparable to that reported here [24, 38]. Thus, further investigations into the cFab format could be warranted.
The scTCR variant has the advantage of being expressed as a single polypeptide chain and contains fewer disulfide bonds. However, this format is often prone to aggregation, and mutational assessment has pointed to three areas of critical importance for scTCR stability, namely amino acids in the Vα/Vβ domain interface, the Vβ HV4 region and surface residues normally shielded by the constant domains that become solvent exposed in the truncated form [22, 24, 38]. Lessons learned from these studies allow for rational protein stability engineering, which was carried out for the TCR380 used in this paper, which has a suboptimal Vα/Vβ domain interface [21]. Specifically, two mutations, L50PTRAV and L103FTRBV, were introduced to form the triad shown to stabilize the Vα/Vβ interface, as L50TRBV was already present. These mutations enable formation of a hydrophobic triad often found at the VH/VL interface of the more stable antibody V domains [22]. In addition, we introduced a proline in position 2 of the linker (L2PLinker), as we have previously shown that this increases the thermostability of a scTCR [17]. In line with this observation, only the stabilized scTCR variant gave good yields of correctly folded monomeric protein. One would expect the stabilized molecule to have the highest thermal resistance [17, 39]. However, both DSF and intrinsic fluorescence spectroscopy used for thermal assessments showed that the scTCR wt380 exhibited a higher Tm than s380. Still, the Vα/Vβ domain interface was engineered to reduce aggregation propensity [40, 41], and the necessity for stability engineering in order to obtain high quality SPR data was demonstrated by the increased binding response observed for scTCR s380 compared to wt380. In SPR, the fraction of functional protein in the injected sample directly affects the estimated affinity values. Our data point to the value obtained for scTCR s380 to best reflect the true monomeric binding affinity, as this sample contains a larger functional fraction.
In a recent study, we employed this version of the stabilized scTCR s380, as well as a stabilized scTCR version of the HLA-DQ2.5:DQ2.5-glia-α2-reactive TCR364, scTCR s364, to evaluate the fine-specificities of the TCRs [21]. By use of these stabilized scTCRs we could carefully evaluate cross-reactivity to highly similar pMHC complexes.
In summary, we demonstrate that RosettaBlueTM expression without IPTG induction results in the overall highest soluble periplasmic expression of all TCR formats tested. Under these conditions, we observed heterodimeric protein formation, and in particular formation of the cFab, and to a smaller extent the dsTCR. To our knowledge, this is the first time successful soluble periplasmic expression of heterodimeric TCRs is reported. Importantly, the periplasmic expression system used herein is compatible with high-throughput screening of TCR variants using 0.5 ml 96-well cultures. Periplasmic samples from these cultures can be analyzed by methods such as ELISA and western blotting. The stabilized version of the scTCR format was expressed in a functional yield that allowed for adequate TCR-pMHC interaction studies with SPR.
Materials and methods
Construction of TCR formats
The cloning of the V genes of the T-cell clone TCR380.E48 into the soluble expression vector pFKPEN [20] to create the scTCR constructs has been described before [21]. The pFKPEN vector is a derivative of the lacPO-based expression vector pHOG21 [33], but has been modified to constitutively express the periplasmic chaperone FkpA for efficient folding of heterologous proteins [20]. The cFab construct was generated by NcoI/HindII and MluI/NotI digestion of pFKPEN-VαβscTCRwt380 followed by sub-cloning of Vα and Vβ into the pFAB-Display phagemid vector on compatible RE sites [26]. The resulting cFab segment was then moved into pFKPEN as a NcoI/SfiI segment creating the pFABEFN-VαCH1-VβCκ-cFab380. The ct-dsTCR construct [13] was generated from mRNA previously isolated from TCC380.E48 using random hexamer primers (Promega) and SuperScriptII reverse transcriptase. Following RNaseH treatment and cDNA precipitation, entire TRA and TRB chains were retrieved using the gene-specific primers TRAV_NcoI_fw/TRA_Spe_rv and TRBV_Mlu_fw and TRB_Sfi_rv, respectively. The non-annealing tails of TRA_Spe_rv and TRB_Sfi_rv encode the antibody-derived C-terminal disulfide bridge. RE digested PCR products were subcloned into the RE digested pFABEFN vector generating the pFABEFN-VαCα-VβCβ-Ct-dsTCR380 vector. The C domain of the TRB chain contains a free cysteine which was removed by QuikChange mutagenesis using TRBC_C85.1A_fw and TRBC_C85.1A_rv primers to avoid aberrant disulphide bond formation. The ct-dsTCR vector generated above was used as a template to generate the dsTCR construct in a step-vise manner. First, non-native cysteines were introduced into TRAC and TRBC by QuikChange mutagenesis using the TRAC_T84C_fw/TRAC_T84C_rv and the TRBC_S79C_fw/TRBC_S79C_rv primer sets. Next, to abolish expression of the antibody-derived cysteine-containing tail of the ct-dsTCR, a stop codon was inserted immediately after TRAC using the TRAV_Nco_fw/dsTRA_Spe_rv primers. Finally, the cysteine-containing tail of TRBC was substituted for a read-through sequence leading into the C-terminal c-myc and His6-tags using the TRB_Mlu_fw/TRB_Sfi_v2_rv primers.
Soluble periplasmic expression
Expression constructs were transformed into E. coli XL1-Blue and RosettaBlueTM host strains. Small-scale expression was performed as follows; cells were inoculated from glycerol stocks into 5 ml 1x LBTAG medium (1x LB medium supplemented with 100 μg/ml ampicillin, 20 μg/ml tetracycline and 0.1M glucose) or 1x LBTACG medium (1x LB medium supplemented with 100 μg/ml ampicillin, 20 μg/ml tetracycline 30 μg/ml chloramphenicol and 0.1 M glucose) for XL1-Blue and RosettaBlueTM strains, respectively. The cultures were grown at ON/37°C/220rpm, followed by re-inoculation to an OD600nm of 0.025 and grown at 37°C/220rpm to an OD600nm of 0.6–0.8. The cultures were spun down at 3220xg/30min and resuspended in 1x LBTA or 1x LBTAC medium either with or without 0.1 mM IPTG for induction, followed by incubation at ON/30°C/220rpm. Cells were pelleted by centrifugation at 3220xg/30min followed by cellular fractionation into medium, periplasmic and cytosolic fractions as described [20]. Large-scale expression (1 L cultures) was performed on the scTCR wt380, scTCR s380, cFab 380 and dsTCR 380 constructs in E. coli RosettaBlueTM host strain without IPTG induction.
Periplasmic fractions from large-scale expression were purified by IMAC on HisTrap HP (GE Healthcare) as described [20]. The eluted fractions were pooled and concentrated by Amicon Ultra (Millipore) followed by size exclusion chromatography (SEC) on a HiLoad 26/600 Superdex200 column (GE Healthcare). A final chromatography step of the samples on either ResourceQ or Superdex75 10/300 GL (both GE Healthcare) columns was performed when needed. The monomeric scTCR wt380 and scTCR s380 molecules or the heterodimeric dsTCR and cFab molecules were pooled and concentrated as before. SEC was performed using PBS with 300 mM NaCl and protein concentration was determined using the MW and extinction coefficient of each individual protein (Denovix DS-11+ Spectrophotometer). Analytical gelfiltration was performed using Superdex 200 Increase 3.2/300 column (GE Healthcare) run in PBS with 300 mM NaCl.
SDS-PAGE and western blot analysis
For western blot analysis of medium, periplasmic and cytosolic fractions, cell cultures were measured by OD600nm and normalized prior to fractionation. 15 μl of each fraction was mixed with loading buffer, heated 5 min at 95°C and run on a Criterion XT Precast 4–12% SDS-PAGE gel (Bio-Rad). Following semi-dry blotting, membranes were blocked with PBS with 4% (w/v) skim milk powder and detected using anti-Histidine tag-HRP antibody (1:10.000, AbD SeroTec, mouse monoclonal, clone AD1.1.10).
For analysis of purified TCRs, 2 μg each of sample was mixed BOLTTM LDS sample buffer, heated 5 min at 95°C before separation on 12% NUPAGE BT gels in BOLTTM MES SDS running buffer (reagents from Novex) along with Spectra prestained multicolor broad-range ladder (Thermo Scientific). Gels were stained with coomassie gel stain. Samples were reduced using DTT as indicated in the figures and figure legends.
Expression of pMHC and purification
Recombinant HLA-DQ2.5 with the gluten-derived peptide DQ2.5-glia-α1a (QLQPFPQPELPY, underlined 9mer core sequence) or CLIP2 (MATPLLMQALPMGAL) coupled to the N-terminus of the HLA-DQ2.5 β-chain via a thrombin cleavable linker peptide was expressed in insect cells using a baculovirus expression vector system as previously described [42, 43]. Soluble, recombinant pMHC was affinity purified using the 2.12.E11 antibody specific for HLA-DQ2 (mouse monoclonal, clone 2.12.E11 [44]). After site-specific biotinylation using BirA (Avidity) as previously described [42], pMHCs were purified using Superdex200 GL10/300 run in PBS.
Differential scanning fluorimetry (DSF)
Protein stability of the purified monomeric scTCRs was measured by DSF using a LightCycler 480 II RT-PCR machine (Roche). SYPRO Orange (Sigma) was used at a 1:1000 dilution and protein concentration at 0.1 mg/ml in a volume of 25 μl. Samples were run in triplicates in 96-well Lightcycler 480 Multiwell Plate. The RT-PCR machine was programmed to ramp the temperature from 20°C to 90°C after a stabilization period for 10 min at 20°C. Data were collected every 0.5°C using the 465 nm excitation and 580 nm emission filters. Data transformation and analysis was performed using the DSF Analysis protocol essentially as described [45].
Fluorescence spectroscopy
The thermostabilities of scTCRs were determined from measurements of intrinsic tryptophan fluorescence during exposure to a temperature gradient from 20°C to 90°C at 1°C/min. Measurements were done in a Jasco-8500 spectrofluorometer equipped with a Peltier temperature control unit using 3 μM proteins in PBS with 300 mM NaCl. 300 μL sample was placed in a 5x5 mm quartz cuvette (HellmaAnalytics). The sample was stirred with a magnetic bead at 150 rpm during the entire measurement. Fluorescence was measured at 1°C intervals during the gradient. The sample was excited at 295 nm and emission measured at 350 nm with medium sensitivity of the photo multiplier (PMT voltage 385V). A marked increase in fluorescence was interpreted as a transition from native to denatured protein. Background was subtracted using the spectra analysis software (Jasco). The relative fluorescence intensity was plotted versus temperature for determination and comparison of Tm using Graphpad Prism 7.
Surface plasmon resonance (SPR)
SPR was performed using a Biacore T200 instrument (GE Healthcare). Neutravidin (10 μg/ml in acetate buffer pH 4.5) was immobilized on a CM3 Series S sensor chip by amine coupling to 1000 resonance units (RU), followed by capture of approximately 100 RU of HLA-DQ2.5:DQ2.5-glia-α1a or HLA-DQ2.5:CLIP2. A 2 fold dilution series ranging from 40 μM to 2.5 μM of soluble scTCR wt380 or scTCR s380 were injected over the HLA-DQ2.5:DQ2.5-glia-α1a-coated surface, while 20, 5 and 0 μM were injected over the HLA-DQ2.5:CLIP2-coated surface. All experiments were run at a flow rate of 30 μl/min at 25○C using single cycle kinetics method (data collection rate 10 Hz). In all experiments, data were zero-adjusted and the Neutravidin reference flow cell value subtracted before analysis using the T200 Evaluation software. A 1:1 Langmuir binding model was used for determination of KD (RI set to constant).
Supporting information
S1 Fig. Amino acid sequences of the TCR constructs.
The amino acid sequence of (A) scTCR wt380, (B) scTCR s380, (C) dsTCR 380, (D) ct-dsTCR 380 and (E) cFab 380. In all variants the leader sequence is highlighted in light grey, variable domains in white, linker in teal and c-Myc and His6 tags in violet. The introduced stabilizing mutations of scTCR s380 (B) is highlighted in red, while stabilizing amino acids already present in the sequence are highlighted in dark red. The TCR constant domains of the dsTCR (C) and ct-dsTCR (D) are highlighted in dark grey and the cysteine residues forming the introduced disulfide bridge are highlighted in pink. The antibody constant of the cFab (D) domains are highlighted in turquoise.
S2 Fig. Expression characteristics of the recombinant TCR formats.
Representative western blot showing the expression profile of the TCR formats. E. coli RosettaBlueTM expression cultures were normalized and fractionated into; M, medium; P, periplasmic; C, cytosolic fractions before analysis by western blots (n = 3–4). All samples were detected with anti-His-HRP antibody. Expression of scFv anti-phOx was included as positive control.
S3 Fig. Original uncropped western blots.
Fig 2A are composed of the following three (A, B, C) uncropped original western blots.
S1 Table. Oligonucleotides.
The authors would like to thank Sivaganesh Sathiaruby for excellent technical assistance and Elin Bergseng for providing soluble, recombinant pMHC. We would like to thank Jens Preben Morth for access to the biophysical characterization platform at the Norwegian Center for Molecular Medicine (NCMM).
- 1. Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V, Bianchi FC, et al. Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Science translational medicine. 2013;5(197):197ra03. pmid:23926201.
- 2. Raman MC, Rizkallah PJ, Simmons R, Donnellan Z, Dukes J, Bossi G, et al. Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy. Scientific reports. 2016;6:18851. pmid:26758806; PubMed Central PMCID: PMC4725365.
- 3. June CH, Warshauer JT, Bluestone JA. Is autoimmunity the Achilles' heel of cancer immunotherapy? Nat Med. 2017;23(5):540–7. pmid:28475571.
- 4. Sharma P, Kranz DM. Recent advances in T-cell engineering for use in immunotherapy. F1000Research. 2016;5. pmid:27703664; PubMed Central PMCID: PMC5031132.
- 5. Molloy PE, Sewell AK, Jakobsen BK. Soluble T cell receptors: novel immunotherapies. Curr Opin Pharmacol. 2005;5(4):438–43. Epub 2005/06/09. pmid:15939669.
- 6. Schmitt TM, Aggen DH, Ishida-Tsubota K, Ochsenreither S, Kranz DM, Greenberg PD. Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro. Nat Biotechnol. 2017;35(12):1188–95. pmid:29106410; PubMed Central PMCID: PMC5722674.
- 7. Maynard J, Petersson K, Wilson DH, Adams EJ, Blondelle SE, Boulanger MJ, et al. Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity. Immunity. 2005;22(1):81–92. Epub 2005/01/25. pmid:15664161.
- 8. Rudolph MG, Stanfield RL, Wilson IA. How TCRs bind MHCs, peptides, and co-receptors. Annu Rev Immunol. 2006;24(1):419–66. pmid:16551255
- 9. Pecorari F, Tissot AC, Pluckthun A. Folding, heterodimeric association and specific peptide recognition of a murine alphabeta T-cell receptor expressed in Escherichia coli. Journal of molecular biology. 1999;285(4):1831–43. Epub 1999/01/26. pmid:9917415.
- 10. Chang HC, Bao Z, Yao Y, Tse AG, Goyarts EC, Madsen M, et al. A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments. Proceedings of the National Academy of Sciences of the United States of America. 1994;91(24):11408–12. Epub 1994/11/22. pmid:7972074; PubMed Central PMCID: PMC45240.
- 11. Willcox BE, Gao GF, Wyer JR, O'Callaghan CA, Boulter JM, Jones EY, et al. Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding. Protein science: a publication of the Protein Society. 1999;8(11):2418–23. Epub 1999/12/14. pmid:10595544; PubMed Central PMCID: PMC2144200.
- 12. Chung S, Wucherpfennig KW, Friedman SM, Hafler DA, Strominger JL. Functional three-domain single-chain T-cell receptors. Proceedings of the National Academy of Sciences of the United States of America. 1994;91(26):12654–8. Epub 1994/12/20. pmid:7809095; PubMed Central PMCID: PMC45497.
- 13. Boulter JM, Glick M, Todorov PT, Baston E, Sami M, Rizkallah P, et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein engineering. 2003;16(9):707–11. Epub 2003/10/16. pmid:14560057.
- 14. Weber S, Traunecker A, Oliveri F, Gerhard W, Karjalainen K. Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor. Nature. 1992;356(6372):793–6. pmid:1315417.
- 15. Novotny J, Ganju RK, Smiley ST, Hussey RE, Luther MA, Recny MA, et al. A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties. Proceedings of the National Academy of Sciences of the United States of America. 1991;88(19):8646–50. pmid:1924326; PubMed Central PMCID: PMC52566.
- 16. Liddy N, Molloy PE, Bennett AD, Boulter JM, Jakobsen BK, Li Y. Production of a soluble disulfide bond-linked TCR in the cytoplasm of Escherichia coli trxB gor mutants. Mol Biotechnol. 2010;45(2):140–9. Epub 2010/02/10. pmid:20143183.
- 17. Gunnarsen KS, Kristinsson SG, Justesen S, Frigstad T, Buus S, Bogen B, et al. Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display. Scientific reports [Internet]. 2013 3557450]; 3:[1162 p.]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23362461 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557450/pdf/srep01162.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557450/pdf/srep01162.pdf.
- 18. Maynard J, Adams EJ, Krogsgaard M, Petersson K, Liu CW, Garcia KC. High-level bacterial secretion of single-chain alphabeta T-cell receptors. Journal of immunological methods. 2005;306(1–2):51–67. pmid:16198365.
- 19. Qiao SW, Raki M, Gunnarsen KS, Loset GA, Lundin KE, Sandlie I, et al. Posttranslational modification of gluten shapes TCR usage in celiac disease. Journal of immunology. 2011;187(6):3064–71. pmid:21849672.
- 20. Gunnarsen KS, Lunde E, Kristiansen PE, Bogen B, Sandlie I, Løset GA. Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA. BMC Biotechnol [Internet]. 2010 2834602]; 10:[8 p.]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20128915 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834602/pdf/1472-6750-10-8.pdf.
- 21. Gunnarsen KS, Høydahl LS, Risnes LF, Dahal-Koirala S, Neumann RS, Bergseng E, et al. A TCRalpha framework-centered codon shapes a biased T cell repertoire through direct MHC and CDR3beta interactions. JCI insight. 2017;2(17). pmid:28878121.
- 22. Richman SA, Aggen DH, Dossett ML, Donermeyer DL, Allen PM, Greenberg PD, et al. Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain ValphaVbeta fragments. Molecular immunology. 2009;46(5):902–16. pmid:18962897; PubMed Central PMCID: PMC2666936.
- 23. Løset GÅ, Lunde E, Bogen B, Brekke OH, Sandlie I. Functional phage display of two murine α/β T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance. Protein Eng Des Sel. 2007;20(9):461–72. pmid:17925331
- 24. Lunde E, Loset GA, Bogen B, Sandlie I. Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins. BMC Biotechnol. 2010;10:61. Epub 2010/08/26. doi: 1472-6750-10-61 [pii] 10.1186/1472-6750-10-61. pmid:20735812; PubMed Central PMCID: PMC2936418.
- 25. Petersen J, Montserrat V, Mujico JR, Loh KL, Beringer DX, van Lummel M, et al. T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease. Nature structural & molecular biology. 2014;21(5):480–8. pmid:24777060.
- 26. Løset GA, Lunde E, Bogen B, Brekke OH, Sandlie I. Functional phage display of two murine alpha/beta T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance. Protein engineering, design & selection: PEDS. 2007;20(9):461–72. Epub 2007/10/11. doi: gzm044 [pii] pmid:17925331.
- 27. Steiner D, Forrer P, Pluckthun A. Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J Mol Biol. 2008;382(5):1211–27. pmid:18706916.
- 28. Welschof M, Terness P, Kipriyanov SM, Stanescu D, Breitling F, Dorsam H, et al. The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody. Proceedings of the National Academy of Sciences of the United States of America. 1997;94(5):1902–7. pmid:9050877; PubMed Central PMCID: PMC20015.
- 29. Petersen J, Kooy-Winkelaar Y, Loh KL, Tran M, van Bergen J, Koning F, et al. Diverse T Cell Receptor Gene Usage in HLA-DQ8-Associated Celiac Disease Converges into a Consensus Binding Solution. Structure. 2016;24(10):1643–57. pmid:27568928.
- 30. Broughton SE, Petersen J, Theodossis A, Scally SW, Loh KL, Thompson A, et al. Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease. Immunity. 2012;37(4):611–21. pmid:23063329.
- 31. Davis MM, Boniface JJ, Reich Z, Lyons D, Hampl J, Arden B, et al. Ligand recognition by alpha beta T cell receptors. Annual review of immunology. 1998;16:523–44. Epub 1998/05/23. pmid:9597140.
- 32. Cole DK, Pumphrey NJ, Boulter JM, Sami M, Bell JI, Gostick E, et al. Human TCR-binding affinity is governed by MHC class restriction. Journal of immunology. 2007;178(9):5727–34. Epub 2007/04/20. doi: 178/9/5727 [pii]. pmid:17442956.
- 33. Kipriyanov SM, Moldenhauer G, Little M. High level production of soluble single chain antibodies in small-scale Escherichia coli cultures. J Immunol Methods. 1997;200(1–2):69–77. pmid:9005945.
- 34. Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nature biotechnology. 2004;22(11):1399–408. Epub 2004/11/06. pmid:15529165.
- 35. Simmons LC, Reilly D, Klimowski L, Raju TS, Meng G, Sims P, et al. Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies. Journal of immunological methods. 2002;263(1–2):133–47. pmid:12009210.
- 36. Robinson MP, Ke N, Lobstein J, Peterson C, Szkodny A, Mansell TJ, et al. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nat Commun. 2015;6:8072. pmid:26311203; PubMed Central PMCID: PMC4560801.
- 37. Lebowitz MS, O'Herrin SM, Hamad AR, Fahmy T, Marguet D, Barnes NC, et al. Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses. Cellular immunology. 1999;192(2):175–84. pmid:10087186.
- 38. Mazor Y, Van Blarcom T, Mabry R, Iverson BL, Georgiou G. Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat Biotechnol. 2007;25(5):563–5. pmid:17435747.
- 39. Shusta EV, Holler PD, Kieke MC, Kranz DM, Wittrup KD. Directed evolution of a stable scaffold for T-cell receptor engineering. Nature biotechnology. 2000;18(7):754–9. pmid:10888844.
- 40. Nieba L, Honegger A, Krebber C, Pluckthun A. Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein engineering. 1997;10(4):435–44. pmid:9194169.
- 41. Bailey LJ, Sheehy KM, Dominik PK, Liang WG, Rui H, Clark M, et al. Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination. J Mol Biol. 2017. pmid:29273204.
- 42. Quarsten H, McAdam SN, Jensen T, Arentz-Hansen H, Molberg O, Lundin KE, et al. Staining of celiac disease-relevant T cells by peptide-DQ2 multimers. Journal of immunology. 2001;167(9):4861–8. Epub 2001/10/24. pmid:11673490.
- 43. Fallang LE, Roh S, Holm A, Bergseng E, Yoon T, Fleckenstein B, et al. Complexes of two cohorts of CLIP peptides and HLA-DQ2 of the autoimmune DR3-DQ2 haplotype are poor substrates for HLA-DM. Journal of immunology. 2008;181(8):5451–61. Epub 2008/10/04. pmid:18832702.
- 44. Viken HD, Paulsen G, Sollid LM, Lundin KE, Tjonnfjord GE, Thorsby E, et al. Characterization of an HLA-DQ2-specific monoclonal antibody. Influence of amino acid substitutions in DQ beta 1*0202. Human immunology. 1995;42(4):319–27. pmid:7558917.
- 45. Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc. 2007;2(9):2212–21. Epub 2007/09/15. doi: nprot.2007.321 [pii] pmid:17853878.


text:
The Oct. 8 webinar, "Healthier, Safer and More Engaged: Your Guide to Creating Total Worker Health," will address safety and health challenges faced by small and mid-sized manufacturers. CNA and NIOSH are co-hosting the event.
CNA is celebrating the fourth annual Manufacturing Day by co-hosting a webinar with NIOSH on Oct. 8 titled "Healthier, Safer and More Engaged: Your Guide to Creating Total Worker HealthTM," which will address challenges faced by small and mid-sized manufacturers to keep employees safe, healthy, and productive in today's economy. Whether the government shutdown affects NIOSH's ability to participate is unclear.
More than 600 manufacturers hosted open houses, public tours, career workshops, and other events in connection with the Oct. 4 Manufacturing Day, according to CNA, an insurance provider. The free webinar will take place at 3 p.m. Eastern time.
"At CNA, we recognize that manufacturing plays a critical role in the U.S. economy. That is why we work together with key members of the industry to promote Manufacturing Day," said Steve Earley, vice president, Manufacturing, CNA. "Through programs like the webinar, we hope to attract attention and new talent to manufacturing while helping support the ongoing prosperity of the whole industry."
Manufacturing Day is co-produced by several organizations, including Fabricators & Manufacturers Association, International® (FMA), and the National Association of Manufacturers. CNA has been the preferred business insurance provider of the FMA for more than 20 years.</
IndustrySafe Safety Software's comprehensive suite of modules help organizations to record and manage incidents, inspections, hazards, behavior based safety observations, and much more. Improve safety with an easy to use tool for tracking, notifying and reporting on key safety data.
IndustrySafe's Dashboard Module allows organizations allows you to easily create and view safety KPIs to help you make informed business decisions. Our best of breed default indicators can also save you valuable time and effort in monitoring safety metrics.
IndustrySafe's Observations module allows managers, supervisors, and employees to conduct observations on employees involved in safety critical behavior. IndustrySafe's pre-built BBS checklists may be used as is, or can be customized to better suit the needs of your organization.
A near miss is an accident that's waiting to happen. Learn how to investigate these close calls and prevent more serious incidents from occurring in the future.
When it comes to safety training, no matter the industry, there are always questions regarding requirements and certifications. We've put together a guide on key safety training topics, requirements for certifications, and answers to common FAQs.


text:
Hi guys, do you looking for huge house plans. May these some photographs for your perfect ideas, whether these images are inspiring photos. Hopefully useful. Comparing medicare plans can save bundle, Preventative care services some home health visits part outpatient prescription drugs third all medicare beneficiaries currently million seniors make choice bundle their under single plan premium.
Navigate your pointer, and click the picture to see the large or full size photo. If you think this collection is useful to you, or maybe your friends you can hit like/share button, so other people can inspired too. Here there are, you can see one of our huge house plans gallery, there are many picture that you can browse, remember to see them too.
Your pool facet would look nice when you've got a large superior garden with landscapes. Large folding glass that opens onto a big deck with pool can be a unbelievable design. The design of a contemporary house aims to mirror the current-day style and taste and often features the newest innovations and assets. A number of the latest developments relate to making a property extremely energy-efficient. Make a point to incorporate the newest technologies and get the architect to fit these in. The crowded and troublesome nature of dwelling in the city make people suppose to dwell with a peaceful life proper by the sea. This design is absolutely good for contemporary homes within the seashore because you possibly can overlook the sea and the sea air can simply enters, providing you with a recent feeling everyday. Others do actually prefer to stay their entire life close to the sea in due to its peaceful and pure ambiance.
Modern kitchen and bathrooms are quite straightforward to choose now, and due to the extensive range, it shouldn't be a problem to discover a set-up to fit your particular person tastes. There are various fashionable beach homes designs you could choose provided by real property sellers. Look for attention-grabbing interiors to swimsuit your entire family, in quite a lot of modern homes the extra formal rooms, such because the dwelling and dining rooms are accomplished away with, and of their place are giant, open plan communal areas. Some house buyers bought seashore properties to have a vacation house throughout summer time. So plan your trendy seashore house design appropriately and select the designs that suit in your character and life-style. The interior design itself would truly create varied expressions and reveal your space sense and the exterior design would present your character and lifestyle. After considering the main development features, it also helps to devise an up-to-date floor plan to accommodate the lifestyle of modern families.
Modern seashore houses designs often have long sloping site and double height structure to the seashore. That is why, modern beach houses become very fashionable and have excessive demands to dwelling consumers. If designed to a high customary, an energy-environment friendly home has the potential to be at least 10% to 20% more effective and saves on vitality consumption, sources, and affords decrease utility payments of the lengthy-time period. If trying to create a house that is really energy-efficient then you can have a look at having rooftop solar panels installed, which when you consider the government supplied incentives (rebates, tax credit, net-metering), does value too much. The design technique of a contemporary designed house is often a very artistic process and follows a number of key design principles. A house with long kitchen dining house also can provides magnificence in the general inside design. 165,000. Obviously, despite the fact that the fee per sq. foot is considerably larger, the general cost to construct is significantly less.
The lifetime worth of a home considers the price of maintaining the house over the years. The small house is making an enormous comeback. What do you actually save by constructing a small residence? While luxury seaside home are those costly ones which might be normally made as an everyday house. Luxury houses are sometimes designed with an open kitchen house which is usually positioned subsequent to the principle family room. A seashore residence often connotes one of two ideals, either a gateway cottage or a luxury beach home roost. Considered one of the most typical modern seaside house designs is a home with extraordinary water view. For the do-it-yourself "professional", the selection of a smaller house is a particularly wise one. The primary features of a contemporary residence are sometimes clear and simple, and from this almost blank canvas it is feasible so as to add a contact of personality by including in colorful fabrics, furnishings and artwork work. The perfect house design is usually styled with clean surfaces and clear traces, with the decoration tone down and painted in light colors.
Should you loved this post and also you wish to receive guidance relating to tudor house model template generously check out our own internet site.
Below are 8 top images from 25 best pictures collection of huge house plans photo in high resolution. Click the image for larger image size and more details.


text:
That's okay, I know how to turn my washing machine into a centrifuge if need be.
The more I look at those eyebrows the more they look like they have been MSpainted into the picture.
As a neuroscientist I have to disagree with the perception that anyone is doing mathematical modeling of cognitive intelligence, yet; intelligence as an economist defines it, yes, but economists are worlds away from actual cognition.
Hrm...Impersonator, fan or the nutjob himself. I suppose time will tell.
If Luna was a furry, she'd sex humans and scream "BEASTIALITY!" at the top of her lungs at inopportune times.
I like the Luna one. She is a good one.
"Stop talking to yourself. You don't like you any better than anyone else who knows you."
So I mocked a serial killer? Great, that's certainly one way to start my morning off.
One day I realized life was pointless. I've been celebrating ever since.
There's beauty in everything so lets destroy it all together.
Also, unlike the other prominent cannibals in the news, this guy hasn't been caught yet. Apparently he is on the loose in France.http://www.dailymail.co.uk/news/article-2153370/Revealed-Victim-Canadian-porn-star-cannibal-Chinese-gay-lover-police-reveal-murderer-run-France--dressed-woman.html
Fuck, supposedly there is a video of him killing his bf kicking about.


text:
Outsourcing and Consulting Services
If it's outside your scope, it's probably within ours. When your business needs extend beyond your core competencies, let the industry's leader in outsourcing and consulting services help you regain your focus. At Kelly Services, our world revolves around your business.
Kelly Outsourcing and Consulting Group (KellyOCG®) offers customizable outsourcing and consulting services to address your every HR need. KellyOCG is a global leader for innovative talent management solutions in the areas of:
- Recruitment Process Outsourcing (RPO)
- Business Process Outsourcing (BPO)
- Contingent Workforce Outsourcing (CWO)
- Career Transition & Executive Coaching
We can deliver consulting services for strategic workforce planning and labor market analysis that enable data-driven talent management. We can partner with your HR department to develop recruiting programs, staffing models and training programs. We can manage your contingent workforce in a vendor neutral environment, as well as provide operational management of entire departments while you focus on what matters most - your core business.
With more than 70 years of experience, we provide the expertise, stability, and global resources to meet youimr specific needs, and we are ready to work with you.
Providing specialty services
We're proud to be a global specialty talent solutions provider to meet the needs of job seekers and employers.
|Life Sciences
|Watch Industry
|Food Industry
|Hotel & Catering
Our solution approaches
Thanks to our local and global network, you get direct access to our talent pool comprising qualified experts with different career levels. Find your appropriate solutions here or let us consult you:
Mandate Service
Permanent Placement
Outsourcing and Consulting Services
Recruitment Process Outsourcing
Temporary Placement
Project Resourcing
Business & Professional Services


text:
- From the Informant, an audio report on how Justice Anthony Kennedy might rule in the California prison-overcrowding case currently before the Supreme Court. Kennedy is considered to be the swing vote.
- Oakland Focus posts this video of Al Davis out exhorting the Raiders' faithful.
- Stop me if this is hyperbole, but I think the Marin Journal's Underbelly blog may be the most clever thing on the entire Internet.
Sign up for our newsletter.


text:
- Roundball Recap: Kansas - Most rational fans assumed Oklahoma would probably not come out on top against Kansas in Lawrence. While the Sooners didn't beat the Jayhawks, the overall play of the Oklahoma offense had to leave you with optimism. The Sooners were flawed, yes. However, through all the turnovers, missed free throws, ill-advised shots and Kansas runs, you … Continue reading "Roundball Recap: Kansas"
- Three Pointer: Kansas - Sad about our college football season ending? Me too. But thankfully we have a good basketball team to numb the pain. The Sooners are 11-1, ranked 23rd in the AP and are incredibly fun to watch. This is a team that is red hot entertaining conference play. Unfortunately, if you are looking for comfort following … Continue reading "Three Pointer: Kansas"
- The Oklahoma Defense Hits Rock Bottom as Sooners beat Kansas 55-40 - KANSAS SCORED FORTY POINTS ON THIS DEFENSE. FORTY!!!!!! I'm honestly so tired of watching this defense squander all time offenses. It's absurd. Mike Stoops wasn't a great defensive coordinator by any means, but it's evident that the issues at defense run much deeper than coaching. This is a non aggressive, poorly tackling and frankly pathetic … Continue reading "The Oklahoma Defense Hits Rock Bottom as Sooners beat Kansas 55-40"
- Weekend Spread: Week 12 - Bobby: 5-3 (46-41) Jamison: 5-3 (42-45) Blake: 5-3 (46-41) Last Week #12 Syracuse v. #3 Notre Dame (ND -9.5) Bobby: I cannot express how badly I want Syracuse to beat Notre Dame. One out of Clemson, Notre Dame or Michigan need to lose for OU to have a window of getting into the College Football … Continue reading "Weekend Spread: Week 12"
- The Schooner Pod: Week 12 - After last week's defensive debacle against Oklahoma State, Bobby and Jamison look at how to fix the unit, the standout Oklahoma performers in the win, take a look at the latest College Football Playoff rankings, examine a wild recruiting weekend in Norman, pick the Week 12 slate and look into Oklahoma basketball's 2-0 start. https://soundcloud.com/schoonerpod/kansas-and-week-12-picks … Continue reading "The Schooner Pod: Week 12"
- Lawrence: The Lost Trip - *hit play for added effect* As some of you know, last season I went to all the home and road games last season. You can read about my travels here. What you'll notice however is the omission of a report from the OU at Kansas game last season; the final stop on the regular season … Continue reading "Lawrence: The Lost Trip"
- Around the Big 12: Week 13 - Last Week: 3-2 Overall: 43-17 Ah shit. Thanksgiving week got the better of me. With that being said, the show must go on with picks, so here is what I thought about the last week of the regular Big 12 season. Baylot @ #12 TCU (TCU won, 45-22. The spread was TCU -25.5.) I … Continue reading "Around the Big 12: Week 13"
- Around the Big 12: Week 12 - Last Week: 5-0 Overall: 40-15 Hey! An undefeated week! Not too bad, especially with how tight the matchups were. Championship November takes a break this week as the contenders are playing the mediocre and downright bad teams of the league. But don't dismiss that 5-5 teams just yet; the fight for a bowl berth is … Continue reading "Around the Big 12: Week 12"
- Game Week: Kansas - So what are everyone's plans for the weekend after last week's madness? Me? I'm going to nap all day, watch some college football, etc. Maybe grill out, relax and go to my date party. Oh wait, I can't do that. I gotta drive five hours to watch Kansas football. Going to every Oklahoma football game … Continue reading "Game Week: Kansas"


text:
Coroutine-based network library
Project description
gevent is a coroutine -based Python networking library that uses greenlet to provide a high-level synchronous API on top of the libev or libuv event loop.
Features include:
- Fast event loop based on libev or libuv.
- Lightweight execution units based on greenlets.
- API that re-uses concepts from the Python standard library (for examples there are events and queues).
- Cooperative sockets with SSL support
- Cooperative DNS queries performed through a threadpool, dnspython, or c-ares.
- Monkey patching utility to get 3rd party modules to become cooperative
- TCP/UDP/HTTP servers
- Subprocess support (through gevent.subprocess)
- Thread pools
gevent is inspired by eventlet but features a more consistent API, simpler implementation and better performance. Read why others use gevent and check out the list of the open source projects based on gevent.
gevent was written by Denis Bilenko.
Since version 1.1, gevent is maintained by Jason Madden for NextThought with help from the contributors and is licensed under the MIT license.
See what's new in the latest major release.
Check out the detailed changelog for this version.
Read the documentation online at http://www.gevent.org.
Post feedback and issues on the bug tracker, mailing list, blog and twitter (@gevent).
Installation and Requirements
Supported Platforms
gevent 1.5 runs on Python 2.7.9 and up, and Python 3.5, 3.6 and 3.7. gevent requires the greenlet library and will install the cffi library by default on Windows. The cffi library will become the default on all platforms in a future release of gevent.
gevent 1.5 also runs on PyPy 7.0 or above. On PyPy, there are no external dependencies.
gevent is tested on Windows, macOS, and Linux, and should run on most other Unix-like operating systems (e.g., FreeBSD, Solaris, etc.)
On Windows using the deprecated libev backend, gevent is limited to a maximum of 1024 open sockets due to limitations in libev. This limitation should not exist with the default libuv backend.
Older Versions of Python
Users of older versions of Python 2 or Python 3 may install an older version of gevent. Note that these versions are generally not supported.
|Python Version
|Gevent Version
|<= 2.7.8
|3.4.0 - 3.4.2
This section is about installing released versions of gevent as distributed on the Python Package Index. For developing with gevent, including running tests, see development. For building gevent from source, including customizing the build and embedded libraries, see Installing From Source.
gevent and greenlet can both be installed with pip, e.g., pip install gevent. Installation using buildout is also supported.
On Windows, macOS, and Linux, both gevent and greenlet are distributed as binary wheels.
You need Pip 8.0 or later, or buildout 2.10.0 to install the binary wheels on Windows or macOS. On Linux, you'll need pip 19 to install the manylinux2010 wheels.
Installing From Source
If you are unable to use the binary wheels (for platforms where no pre-built wheels are available or if wheel installation is disabled), you can build gevent from source. A normal pip install will fall back to doing this if no binary wheel is available. See Installing From Source for more, including common installation issues.
Extra Dependencies
gevent has no runtime dependencies outside the standard library, greenlet, and (on some platforms) cffi. However, there are a number of additional libraries that extend gevent's functionality and will be used if they are available. All of these may be installed using setuptools extras, as named below, e.g., pip install gevent[events].
- events
- Configurable event support using zope.event is highly recommended for configurable event support.
- dnspython
- Enables the new pure-Python resolver, backed by dnspython. On Python 2, this also includes idna. They can be installed with the dnspython extra.
- monitor
- Enhancements to gevent's self-monitoring capabilities. This includes the psutil library which is needed to monitor memory usage. (Note that this may not build on all platforms.)
- recommended
- A shortcut for installing suggested extras together.
- test
- Everything needed to run the complete gevent test suite.
For development information, including installing from git and running tests, see development.
Project details
Release history Release notifications
Download files
Download the file for your platform. If you're not sure which to choose, learn more about installing packages.


text: Well, well, what do we have here... Looks like the real collusion story is yet to be told.. Ukraine, Ukraine, Ukraine? https://thehill.com/opinion/white-h...s-dnc-contractor-solicited-trump-dirt-in-2016 *snip* The boomerang from the Democratic Party's failed attempt to connect Donald Trump to Russia's 2016 election meddling is picking up speed, and its flight path crosses right through Moscow's pesky neighbor, Ukraine. That is where there is growing evidence a foreign power was asked, and in some cases tried, to help Hillary Clinton. In its most detailed account yet, Ukraine's embassy in Washington says a Democratic National Committee insider during the 2016 election solicited dirt on Donald Trump's campaign chairman and even tried to enlist the country's president to help.