Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins
Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidation of phenol-tagged cargo molecules is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepared and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chemistry for the activation of tyrosine residues that extend into solution from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner, and it can extend the reaction chemistry to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The compatibility of several secondary amines and aniline derivatives was evaluated in the coupling reactions, providing important information for substrate design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.
Introduction<!>Results and Discussion<!>Conclusion<!>III. Instrumentation, Reagents, and Sample Analysis<!>Mass Spectrometry of Proteins:<!>UV-VIS measurements:<!>NMR Spectroscopy:<!>HPLC purification:<!>Flow Cytometry Analysis:<!>IV. Protein Sequences and DNA Manipulation<!>DNA and Protein Sequences:<!>Cytosolic expression of recombinant proteins -Liter Scale:<!>Cytosolic Expression of Recombinant Proteins -10-50 mL Scale:<!>Periplasmic expression and purification of Trastuzumab scFv:<!>VI. Tyrosinase-Mediated Oxidative Coupling Reactions of Proteins<!>Standard Tyrosinase-mediated oxidative coupling reaction for subsequent TOF-LCMS analysis:<!>Modification of pAF-MS2 with N-Ac-α-endorphin peptide:<!>VIII. Protein Binding Experiments and Flow Cytometry Analysis<!>Binding of scFv-O.G. 488 Conjugate and negative controls:<!>Binding of Protein-L-AN20GGY-O.G. 488 conjugate to Trastuzumab scFv-treated cells:<!>IX. NMR Spectra
<p>Site-selective protein bioconjugation reactions are critical for the construction of chemical biology probes, targeted delivery agents 1 , and well-defined biomaterials 2,3 . In particular, the conjugation of fluorophores, drugs, and other small molecules to antibodies and other binding proteins has become a standard approach for constructing immunodetection reagents 4 , improving the therapeutic indices of toxic drugs through antibody-drug conjugates 5,6 (ADCs), and directing molecular tools to target antigens 7,8 . These chemistries are also critical for attaching proteins to surfaces in defined orientations, enabling the miniaturization of diagnostics and electronic devices interfaced with living systems via proteins 9,10 . Often, surface cysteine residues 11,12 or unnatural amino acids are used as attachment points 13 , and several approaches have taken advantage of the unique reactivity of the N-terminal positions to create protein conjugates [14][15][16][17] . While these strategies target functional groups that can be installed conveniently in many cases, there remain instances in which the introduction of new nucleophilic residues can lower expression yields or hinder protein function. As an example, the N-termini of immunoglobulins are typically on the same face of the protein as the antigen binding loops, which can limit the utility of these sites as points for surface attachment or large cargo installation.</p><p>The C-terminus can provide another site for modification, though it has historically been more difficult to functionalize. C-terminal decarboxylative alkylation was achieved recently with photoredox chemistry using a water-soluble photocatalyst and by taking advantage of the lower oxidation potential of this site relative to side-chain carboxylates. Intein trans splicing in the most commonly used technique for attaching peptide sequences to C-termini, though this method requires the genetic fusion of bulky constructs and leaves a cysteine residue at the ligation site 18 . As another alternative, the widely used Sortase enzyme uses -LPXTG recognition sequences to link cargo groups bearing a polyglycine (n = 1-5) motifs to protein C-termini after the threonine residue in a LPXTG sequence 19,20 . Butelase 21 , subtiligase 22 farnesyl transferase 23,24 , and SpyTag/SpyCatcher 25,26 , work similarly by recognizing and ligating specific peptide sequences. Tubulin tyrosine ligase 27,28 , and AnkX 29,30 (a bacterial phosphocholine transferase) can also append synthetically modified small molecule substrates to proteins based on peptide recognition sequences. Each of these methods has advantages and disadvantages to consider when choosing a preferred methodology, including the size of the recognition motif, the residual sequence left behind after modification, the required pH and buffer conditions, the synthetic accessibility of the coupling partners, and the kinetics, availability, stability, and selectivity of the enzyme catalyst.</p><p>Our lab has developed a set of oxidative coupling chemistries that involve the reaction of orthoquinone and iminoquinone intermediates with N-terminal proline residues and aniline groups (Figure 1a). 31 This set of techniques can be used to conjugate a wide range of small molecules, nucleic acids, nanoparticles, surfaces, and additional proteins to specific locations on biomolecular substrates. However, two features of this reaction limit its use. First, the use of strong oxidants, such as sodium periodate, can result in collateral oxidation of cysteine and methionine side chains, and even protein-tolerant oxidants like ferricyanide ion need to be removed from the protein solution following the reaction. Secondly, some of the o-quinone precursors require synthetic expertise to make and can be difficult to store due to oxidation by atmospheric oxygen (although the storage issue can be addressed using o-methoxyphenol substrates 32 ). Recently, we reported that the tyrosinase enzyme from the common button mushroom Agaricus bisporus (abTYR) is capable of oxidizing simple phenols attached to cargo molecules to yield o-quinones in situ (Figure 1b). 14 The enzyme relies on two copper ions chelated in a deep active site pocket to transfer an atom from molecular O2 to the ortho-position of a bound phenol (Figure 1c-e). 33,34 The enzyme operates rapidly at 4 °C and room temperature at a mild pH of 6.5, is commercially available, and can be stored at -80 °C in solution for months. It readily oxidizes phenols that are appended with NHS-activated cargo molecules, allowing simple tyramine derivatives to be used as readily available and storable building blocks. Most importantly, in previous work, 14 we and others have found that it leaves native tyrosine residues on proteins untouched if they are within the secondary and tertiary structural elements of folded structure.</p><p>The ability of tyrosinases to generate o-quinones on proteins is well documented, at least at high substrate concentrations. Tyrosinases and phenol oxidases have been known for decades to mediate crosslinking between proteins in meat 35 , whey 36 , and flour 37 via non-selective tyrosine-tyrosine, tyrosinecysteine, and tyrosine-lysine linkages. More recently, Long and Hedstrom described abTYR-mediated oxidation of multiple tyrosine residues in a hemagglutinin tag (YPYDVPDYA) and similar variants at the C-terminus of E. coli dihydrofolate reductase 38 . Although a hydrazide-functionalized dye could be attached to the oxidized tag, fragmentation of the sequence was a major side reaction. The many reports of tyrosinase-generated o-quinones modifying proteins via different linkages may have been a deterrent against pursuing these species as handles for selective modification in the past. As one example, Montanari et al. specifically sought to suppress o-quinone formation during tyrosinase-mediated oxidation of tyrosines to catechols for conjugation with boronic acids, out of a concern that these species would cause detrimental side reactions 39 . The most successful utilization of abTYR for protein modification to date was by Delft and co-workers, who used it to prime a tyrosine-tagged antibody for strain-promoted Diels-Alder cycloaddition with a cyclooctyne-functionalized drug molecule 40 .</p><p>The field of organic chemistry is filled with examples of o-quinones reacting with phosphines 41 , thiols 34,42 , amines 42 , and other diverse nucleophiles. The challenge then becomes the identification of nucleophiles that can compete with undesired, sometimes intramolecular pathways to afford clean bioconjugates without crosslinking. Here, we report that tyrosine residues encoded on linkers at protein N or C-termini (tyrosine tags or -GGY) can be readily oxidized by abTYR and the smaller, readily expressed, Bacillus megaterium tyrosinase (bmTYR) for subsequent reaction with secondary amine and aniline nucleophiles (Figure 1f). These reactions are demonstrated for a number of different proteins, including an antibody single chain variable fragment and immunoglobulin-binding Protein L. This work expands the range of protein substrates that can be modified using OC techniques, including the modification of Cterminal positions on proteins, allows greater flexibility in the selection of nucleophilic coupling partners, and provides the field with a new tyrosinase with improved reactivity.</p><!><p>These studies began by examining whether the N-terminal N-acyl-tyrosine residue on the αendorphin peptide would be sterically unencumbered enough to be oxidized by abTYR (Figure 2a). This was tested by combining this peptide at 100 µM with MS2 capsids (10 µM, based on monomer concentration) bearing p-aminophenylalanine (paF) residues that were introduced using amber codon suppression 43,44 . In the presence of 83 nM abTYR, clean, nearly quantitative conversion was observed to the expected aniline addition product (Figure 2b). As there are 180 copies of the aniline groups in each capsid, this corresponded to the installation of >170 new peptide groups on the external surface of each assembly in 2 h at room temperature. The identity of the product was confirmed for a small molecule model resulting from the tyrosinase-mediated addition of N-acetyl-L-tyrosine to p-toluidine in D2O with abTYR. The reaction required more tyrosinase (350 nM corresponding to 42 U/mL) and an extended reaction time to proceed in D2O, and turned purple. Direct NMR analysis confirmed that the initially formed 5-anilino-1,2-catechol is subsequently oxidized back to the quinone state, presumably by adventitious oxygen in the solution (see NMR section of Supporting Information). The 5.7 ppm proton signal attached to the 5 carbon of the oxidized tyrosine phenol gradually diminished over the course of several hours as it exchanged with deuterium in solution, consistent with enol tautomerization. As has been previously reported by our group for products generated from o-aminophenols 45 , this species exists as the p-iminoquinone tautomer in solution. The mass of the MS2-peptide conjugates corresponded exactly to the analogous small molecule product.</p><p>It should be noted that acetylation of the α-endorphin N-terminus was essential to the success of the reaction, since a free N-terminal amine would readily attack the proximal o-quinone as it does upon the oxidation of free L-tyrosine during melanin biosynthesis 34 . We have confirmed this pathway for other peptides, and thus we recommend using peptides with acyl or glycine residues before the tyrosine site.</p><p>To test the abTYR OC reaction on a more challenging protein substrate, a -GGY tag was appended to the C-terminus of a single chain variable fragment (scFv) of the Trastuzumab antibody. Trastuzumab is an FDA approved monoclonal antibody for the treatment of HER2(+) breast cancer, 46 and is widely used as a model construct for testing bioconjugation reactions. 19,40,47,48 Trastuzumab is also commonly used in an scFv format, which is of interest because of its improved tissue and tumor penetration relative to full length antibodies 49,50 and its potential to be used for constructing bispecific antibodies. 51 We chose to use an established E. coli periplasmic expression protocol to produce the Trastuzumab scFv with a C-terminal -GGY tag, 52 Figure 3a-c. One potential challenge presented by this substrate is that, of its 15 tyrosine residues, 8 are located on the antigen binding site with their phenol side chains oriented toward the bulk solution. Given the potential for off target oxidation of the internal tyrosine residues, we were gratified to find that the C-terminally -GGY tagged version of the scFv coupled in nearly quantitative yield to aniline (Figure 3c), while a non-tyrosine tagged version was virtually untouched under the reaction conditions (see Supporting Information Figure S1). This substrate was next used for the evaluation of several parameters of the reaction. To ensure meaningful comparisons, abTYR samples were assayed for reactivity by following the oxidation of Ltyrosine and subsequent dopachrome formation 34 with UV/vis spectroscopy before use. Enzyme concentrations in subsequent reactions were adjusted such that the same final U/mL of enzyme were used for each. This was necessary because equal masses of different batches of abTYR were found to vary somewhat in their enzymatic activity. Typically, 1 mg/mL tyrosinase stock solutions had activities between 900 and 1200 U/mL, corresponding to 83 to 111 nM enzyme in a typical 12 U/mL reaction. Varying the concentration of aniline from 25 µM to 750 µM showed that maximum conversion was achieved at a concentration of 150 µM with at least 8 U/mL abTYR (Supporting Information Figure S2). At lower abTYR concentrations, higher concentrations of aniline appeared to inhibit the enzyme, as evidenced by residual un-oxidized starting material in these reactions. The fact that residual uncoupled protein was fully oxidized in reactions with at least 8 U/mL tyrosinase suggests that the coupling partner has a limited time to intercept the o-quinone intermediate before it is quenched by a competing process (likely an internal nucleophile on the protein). In a delayed nucleophile addition experiment, aniline was added to a series of scFv-GGY OC reactions 5, 10, 20, 40, or 60 min after abTYR. This experiment showed that, while the starting protein is completely oxidized in every case, the amount of product formed depends upon how soon after the start of the reaction the coupling partner is added (Supporting Information Figure S3).</p><p>The coupling efficiencies of various piperazines and a racemic N-methyl pyrrolidine were also assessed (Figure 2d and Supporting Information Figures S4 and S5). These proved to be less reactive nucleophiles, and required much higher concentrations to achieve good conversion. Racemic proline-Nmethylamide provided the best conversion in this category of nucleophiles, consistent with our previous findings that proline N-termini are efficient oxidative coupling partners. 14,16,53 To confirm that the abTYR-mediated modification of -GGY tagged Trastuzumab scFv did not perturb the binding activity of this construct, the fluorescent dye Oregon Green 488 (OG 488) was derivatized with an aniline nucleophile and oxidatively coupled onto the scFv (Supporting Information Figure S6a). The reaction proceeded optimally with 25 µM aniline-OG 488 and 12 U/mL abTYR to give the scFv-GGY-OG 488 construct with 84% conversion in 1 h. Flow cytometry showed that this construct could recognize the HER2(+) SK-BR-3 breast cancer cell line selectively over HER2(-) MDA-MB-468 breast cancer cells (Figure 4c).</p><p>The stability of the aniline conjugate to Tyr-tagged scFv was monitored at 4 °C over the course of 7 days in a standard phosphate and NaCl protein storage buffer with 15% glycerol (pH 7.4). The linkage was found to remain intact throughout this period (Supporting Information Figure S7). The addition of 10 mM dithiothreitol (Supporting Information Figure S8) or glutathione (Supporting Information Figure S9) resulted in the formation of thiol adducts to the new aromatic ring, but did not result in cleavage of the linkage between the protein and the small molecule. In the case of glutathione, this further modification occurred quantitatively within 24 h. Moreover, subsequent treatment of the scFv-GGY-aniline-glutathione product with DTT for 24 h showed that the initial glutathione adduct was not released competitively. This indicates that the pre-addition of glutathione could be used to avoid the addition of other thiols in serum, if required (Supporting Information Figure S10). Protein L is an IgG binding protein that recognizes variable regions of human kappa light chains. Discovered in pathogenic Peptostreptococcus magnus, it consists of five binding domains 54 connected in tandem by short 9-10 amino acid linkers 55 (Figure 5a). The avidity of the combined interactions of these five domains allows the protein to achieve a Kd value of 130 nM at pH 8 56 . When expressed recombinantly, cell-wall anchoring domains of the wild type protein are typically omitted, and some truncated versions have only 3 or 4 light chain binding domains. Protein L is routinely used for the purification of scFv constructs 57 and has been employed as a universal flow cytometry marker for cells expressing Chimeric Antigen Receptors (CARs) 58 , which typically utilize scFv segments to recognize their targets. Because Protein L binds to variable light chains without interfering with the antigen recognition loops, it can be used as a "secondary" detection reagent for bound scFv's and IgG's.</p><p>We expressed Protein L in E. coli with flexible, C-terminal -GGY and -GGGGSGGY tags for abTYR-mediated modification with aniline-functionalized OG 488 to create secondary scFv detection reagents. Surprisingly, these constructs were resistant to oxidation by abTYR, most likely because the terminal tyrosine residue does not extend sufficiently from the bulk of the protein structure to reach into the sterically occluded abTYR active site. In an attempt to circumvent this problem, we generated a panel of Protein L variants with C-terminal linkers of various types and lengths preceding the tyrosine tag (Figure 5b and Supporting Information Figure S11 and S12). In addition to the flexible (G4S)1-3 linkers, we extended Protein L with an alpha-helical (EAAAK)2 repeat sequence 59 , a non-hydrogen bonding, rigid (AP)3 repeat 60 , a poly asparagine (N20) sequence, and the C-terminal sequence of the easily-modified Trastuzumab scFv (EIKRTGGY). Additionally, the C-terminal 5 th light chain binding domain was deleted and the native linker between the 4 th and 5 th light chain binding domains was extended with -GGY and -G4SGGY tags. Disappointingly, none of these variants could be oxidized by abTYR.</p><p>We recognized that the steric bulk of the 120 kDa tyrosinase protein likely makes access to the active site difficult for many protein substrates. While in principle it should be possible to generate an abTYR-oxidizable variant by continuing to extend the C-terminal linker, longer linkers increase the risk of the tag interfering with protein function, and are more difficult to install during gene construction, dampening the convenience of the tyrosine tagging approach. We were therefore motivated to express and test the much smaller Bacillus megaterium tyrosinase (bmTYR). This 35.5 kDa protein is robustly expressed in BL21 (DE3) E. coli with a yield of up to 160 mg/L and has a much more accessible active site in comparison to abTYR. Gratifyingly, all tyrosine-tagged Protein L variants exposed to bmTYR were quantitatively oxidized and reacted with 150 µM aniline with over 90% conversion in 1 h, while the nontagged variant was still untouched (Figure 5b, Supporting Information Figure S11 and S12).</p><p>With a solution to Protein L modification in hand, we next performed the bmTYR mediated OC with 25 µM aniline-OG 488 on the -AN20GGY tagged Protein L variant. The desired conjugate was obtained with 87% conversion in 1 h (Figure 6c, Supporting Information Figure S6b). This construct was then applied to HER2(+) (SK-BR-3) or HER2(-) (MDA-MB-468) breast cancer cells that had been pretreated with the non-tyrosine tagged trastuzumab scFv. Only the Protein L construct that had been modified with aniline-OG 488 under the bmTYR-mediated OC conditions was able to label the HER2(+) cells after exposure to the scFv (Figure 6d). Cells without HER2 or samples with a component omitted from the coupling and labeling workflow were untagged.</p><p>It is noteworthy that the substrates presented herein do not have surface-exposed cysteine residues, as we have found that these can also participate in efficient couplings between peptide and protein substrates. This work is reported in a separate publication 61 . If this interferes with the addition of aniline and cyclic amine nucleophiles, we have shown in previous studies 16 that thiol nucleophiles can be conveniently capped through disulfide formation with Ellman's reagent. Following the oxidative coupling reaction, these groups can be removed through brief exposure to an appropriate reducing agent.</p><!><p>In this work, we have expanded the capabilities of oxidative coupling chemistry by showing that tyrosinase enzymes can be used to generate o-quinones on sterically-exposed C or N-terminal tyrosine residues. These groups can then be coupled to aniline or cyclic-amine nucleophiles, which compete favorably with internal nucleophiles in the protein itself. The chemical modification of native amino acids is an appealing approach to protein modification because it avoids the increased complexity and lower expression yields that can be associated with noncanonical amino acid incorporation. Most native amino acid targeting strategies, including the established oxidative coupling approaches, modify chemically distinct groups through the use of carefully calibrated electrophiles added exogenously. Our strategy reverses that common paradigm by unmasking a latent electrophile on the protein substrate with a restrained oxidizing agent, giving the user flexibility in choosing the nucleophilic coupling partner. This shift eliminates the problem of off-target modification by the small molecule, which is difficult to avoid because bioconjugation reactions on proteins typically use a large excess of the small molecule component. The presence of the purified protein substrate at much lower concentrations (nanomolar to low micromolar) minimizes off-target protein-protein coupling as well, yielding cleaner coupling products overall.</p><!><p>Reagents:</p><p>All chemicals were ordered from Sigma Aldrich or ThermoFisher Scientific and used as received unless noted otherwise. Milli-Q H2O was obtained from a Barnstead NANOpure Diamond water purification system. Oregon Green 488 Sucinimidyl ester (5 isomer) was obtained from ThermoFisher Scientific (Product # O6147). A Pierce BCA Assay Kit (Thermo Scientific) was used to determine concentrations of purified protein aliquots.</p><!><p>Time of flight electrospray ionization mass spectrometry (TOF-ESI-MS) of proteins was performed using an Agilent 1260 series liquid chromatography (LC) system with an Agilent 6224 TOF LC-MS system (Santa Clara, CA). For mobile phases, the system utilized Milli-Q H2O with 0.1% (v/v) formic acid (solvent A) and HPLC grade ACN with 0.1% (v/v) formic acid (solvent B). Before submission to analysis, each sample was desalted and passed through a 0.22 µm cellulose acetate centrifugal filter. 2 -10 µL were injected, corresponding to 0.2 to 1.0 µg of protein. The sample was separated on a Proswift RP-4H (monolithic phenyl, 1.0 mm × 50 mm, Dionex) analytical column with a flow rate of 0.30 mL/min and a gradient of 5-100% solvent B in solvent A over 8 minutes. Agilent Mass Hunter Workstation software (Qualitative Analysis Version B.05.00, Build 5.0.519.13, ©Agilent Technologies Inc. 2011) was used to extract total ion chromatograms from raw data and the Maximum Entropy deconvolution algorithm was used to reconstruct the original analyte masses. CSV files of spectra were plotted using the open source Chartograph software (www.chartograph.com). Integration of an unmodified protein sample served as an internal standard for determining conversion.</p><!><p>A NanoDrop 1000 (Thermo) was used to quantify the concentrations of DNA plasmid samples and for measuring protein concentrations of samples in BCA assays. A Varian Cray 50 scan UV-Visible spectrometer was used for measuring the optical densities (O.D.) of cell cultures at 600 nm and for monitoring absorbance of dopachrome at 475 nm for tyrosinase activity assays.</p><!><p>The small molecule thiol coupling product was characterized on a Bruker 900 MHz NMR spectrometer at the at the UC Berkeley QB3 NMR facility. Funds for the 900 MHz NMR spectrometer were provided by the NIH through grant GM68933.</p><!><p>Semiprep HPLC purification (for Aniline-Oregon Green 488) was performed on an Agilent 1260 Infinity series system equipped with a UV-Vis detector and fraction collector, controlled by Open Lab CDS software (©Agilent Technologies Inc. 2011 -2015). Milli-Q H2O and HPLC grade ACN were used as mobile phases. A Phenomenex Gemini C18, 250 x 10.0 mm column was used (5 µm, 110 Å).</p><!><p>Cell samples were diluted 2-3x in PBS and submitted to a BD Biosciences, BD LSR II flow cytometer. Fluorescence excitation at the 488 nm laser line and FITC filter set were used. Data was acquired using BD FACSDiva Software (version 6.2, ©2000) 10,000 events were recorded for each sample. Data was processed using FlowJo TM software (version 10).</p><!><p>Protein gene blocks were ordered from Integrated DNA Technologies, codon-optimized for E. coli expression. Bsa1 cut sites were present at either end of the gene sequence for cloning into the pET28b golden gate entry vector. This vector enabled green / white screening for colonies successfully transformed with plasmids bearing the inserted gene and provided resistance to kanamycin. PCR primers used to make Protein L variants with different C-terminal tags are listed in Supporting Table 2.</p><!><p>PelB</p><!><p>A 10 mL overnight culture of BL21 (DE3) Star E. coli in TB medium with 50 µg/mL kanamycin was inoculated into a 2.8 L trident Erlenmeyer flask with 1 L of sterile Teriffic Broth (TB) medium and kanamycin. The cultures were grown at 37 °C with 220 rpm shaking until O.D. at 600 nm reached 0.6 -0.8. To induce expression, IPTG was added from a 1.0 M stock to a final concentration of 1.0 mM. The cultures were incubated at 37 °C for 18-20 hours. The culture was pelleted via centrifugation (3,200 g, 15 min, 4 °C) and the supernatant was discarded. All steps from this point were performed with cold (4 °C) buffers and on ice. Each pellet was re-suspended in 45 mL of D.I. H2O then pelleted in 50 mL falcon tubes (3,200 g, 15 min, 4 °C). The supernatant was poured off and the pellets were either stored at -80 °C or lysed immediately.</p><p>Each cell pellet (from 0.5 L culture) was re-suspended in 25 mL of lysis buffer (PBS, 20 mM imidazole, pH 7.4 with 0.1 mM Phenylmethylsulfonyl fluoride, 0.05 U/mL benzynase, and 0.5 mg/mL chicken egg white lysozyme). The cells were incubated on ice for at least 30 minutes then lysed by 10 min of sonication (2 sec on, 3 sec off pulses at 60% amplitude with a Fisher Scientific Sonic Dismembrator). The resulting suspension was centrifuged (14,000 g, 30 min, 4 °C) and the supernatant was passed through a 0.2 µm syringe filter. The crude lysate was subject to NiNTA purification using a G.E. Healthcare HisTrap HP 5mL affinity column equilibrated with Buffer A (PBS with 20 mM imidazole, pH 7.4) on an ÄKTA pure FPLC system (G.E. Healthcare). The protein was eluted using a gradient of 0-100% Buffer B (PBS with 400 mM imidazole, pH 7.4) over 20 column volumes. Product-containing fractions were identified using SDS-PAGE and submitted to further purification as needed. Purified protein was exchanged into protein storage buffer (PBS with 15% glycerol) via 4 rounds of spin concentration in 10 kDa MWCO, 15 mL amicon ultra centrifugal filters, diluting with storage buffer before each spin.</p><!><p>Expression and cell pellet processing as above. Pellets were re-suspended in 1.0 mL of lysis buffer and sonicated for 45 sec (5 sec on, 5 sec off pulses at 50% amplitude). Lysates were centrifuged for 10 min at 16,100 g. Supernatant was retained and solids were discarded. For each lysate, 10 µL of HisPur NiNTA resin (Thermo scientific product #88221; capacity of 60 mg His6-protein per mL suspended resin) per mL of cell culture was equilibrated with binding buffer (PBS, 20 mM Imidazole, pH 7.4). The resin was then suspended in the cell lysate and incubated at 4 °C for 1 hour. After binding, resin aliquots were washed 2x with binding buffer, then bound proteins were released with elution buffer (PBS, 80 mM Imidazole, pH 7.4). The purity of eluted proteins was assessed with SDS-PAGE and TOF-LCMS. Proteins were exchanged into protein storage buffer (20 mM Na2HPO4, 150 mM NaCl, in 15% glycerol, pH 7.4) via 4 cycles of centrifugation (14,000 g, 20 min, 4 °C) in 10 kDa MWCO 500 µL amicon ultra spin desalting filters, diluting to 500 µL with protein storage buffer before each cycle.</p><!><p>The following procedure was adapted from a protocol by Rouet et al. 1 . Two finless 4 L Erlenmeyer flasks containing 1 L of sterile Teriffic Broth medium with 50 µg/mL kanamycin each, were inoculated with 10 mL overnight TB medium cultures of BL21 (DE3) Star E. coli cells bearing the PelB -Trastuzumab scFv -GGY or wild type Cterminus in pET28b. The cultures were grown at 37 °C with 220 rpm shaking until O.D. at 600 nm reached 0.6 -0.8.</p><p>To induce expression, IPTG was added from a 1.0 M stock to a final concentration of 0.4 mM. The cultures were then incubated at 28 °C with 200 rpm shaking. After 4 hours, each 1 L culture was divided into two centrifuge bottles and pelleted by centrifugation (3,200 g, 15 min, 4 °C) and the supernatant was discarded. All steps from this point were performed with cold (4 °C) buffers and on ice. Each pellet (from 0.5 L culture) was re-suspended in 25 mL of periplasmic extraction buffer 1 (20% w/v sucrose, 100 mM TRIS-HCl, 1.0 mM EDTA, pH 8.0) and incubated for 30 min. The cells were pelleted by centrifugation (10,000 g, 10 min, 4 °C) using a fixed-angle rotor, and the supernatant was poured off and saved as "periplasmic extract 1". The pellets were each re-suspended in 25 mL of periplasmic extraction buffer 2 (5.0 mM MgCl2) and incubated on ice for 30 min. The cells were again pelleted by centrifugation (10,000 g, 10 min, 4 °C) and the supernatant was poured off and saved as "periplasmic extract 2". The periplasmic extracts were both found to contain scFv by SDS-PAGE. They were combined, concentrated to 50 mL via multiple rounds of centrifugation in 10 kDa MWCO amicon ultra centrifugal filters (4000 g, 20 min, 4 °C), then passed through 0.22 µm PES syringe filters * . The periplasmic extracts were transferred to 3500 Da MWCO dialysis cassettes and incubated in 4 L of PBS overnight with gentle stirring.</p><p>A 5 mL G.E. Healthcare Capto L (resin-bound Protein L) affinity column was equilibrated with Buffer A (50 mM citrate, 400 mM NaCl, pH 6.0). The dialyzed periplasmic extracts were again passed through a 0.22 µm PES syringe filter, loaded onto the Capto L column, and eluted with a gradient of 0-70% Buffer B ** (50 mM citrate, pH 2.5) over 22 column volumes followed by 100%B for 8 column volumes. Fraction collection tubes were pre-filled with 1.0 mL of 1.0 M TRIS HCl buffer at pH 8.0 to neutralize elution buffer *** . scFv typically eluted at 100% buffer B and was pure by TOF-LCMS. 0.8 mg to 1.2 mg per liter of expression media was obtained.</p><!><p>Tyrosinase stocks and activity assay:</p><p>Agaricus bisporus Tyrosinase (abTYR) was obtained from Sigma Aldrich as a lyophilized powder and stored at -20 °C when received. 1 mg/mL stock solutions were prepared in 50 mM phosphate buffer, in 15% glycerol in water, pH 6.5, stored at -80 °C. Bacillus megaterium Tyrosinase was obtained by recombinant expression from E. coli as described above and stored as a stock solution in a similar buffer after purification. Tyrosinase aliquots were thawed on ice shortly before use and the activity of each new stock was assayed by monitoring conversion of L-Tyrosine to dopachrome via UV-Vis as described previously 2 . A stability study 2 has indicated that the activity of abTYR aliquots prepared in this manner is stable over months of storage at -80 °C.</p><!><p>Volumes of each stock solution indicated in the table above were added to an Eppendorf tube with the tyrosinase enzyme added last. Protein concentrations ranged from 1.0 to 4.0 µM. Phosphate concentration was always 20 mM and total reaction volumes and nucleophile concentrations were changed accordingly. The resulting solution was * In some cases, filters clogged very rapidly and an extra centrifugation step (36,000 g, 30 min, 4 °C) was necessary to remove fine particles. ** Make from citric acid rather than sodium citrate to minimize salt concentration from titrating to pH 2.5. High ionic strength promotes Protein L binding. *** 1.0 mL of 1.0 M TRIS-HCl at pH 8 and 5.0 mL of Buffer B brought final solution pH to 5.2. The quenched reaction was subject to four cycles of spin desalting in a 10 kDa MWCO 500 µL amicon ultra centrifugal filter, diluting with 400 µL of Milli-Q H2O before each cycle to remove aniline, and phosphate buffer. The resulting 35-50 µL droplet was passed through a 0.22 µm cellulose acetate centrifugal filter and submitted to TOF-LCMS analysis. This standard procedure was followed unless indicated otherwise.</p><!><p>To a 10 µM solution of pAF-MS2 Expressed as described by Carrico et al. 3 , (concentration based on monomer) in 20 mM phosphate buffer, pH 6.5, was added NAc-alpha-endorphin (obtained from Genscript) to a final concentration of 100 µM. To this was then added abTYR to final concentration of 83 nM and the reaction was allowed to sit at room temperature for 2 h, after which the sample was diluted 2x with 10 mM bis-tris buffer, pH 7.2. Samples were filtered using 0.22 µm cellulose acetate centrifugal filter and analyzed via ESI-TOF MS.</p><!><p>Cell Preparation for scFv binding:</p><p>T-25 flasks with MDA-MB-468 (HER2(-)) cells or SK-BR-3 (HER2(+)) cells were obtained from the Berkeley Cell Culture Facility and grown to 90-100% confluency in a 37 °C incubator with 5% CO2. In preparation for analysis, growth medium was removed, and adherent cells were rinsed with D-PBS. Cells were detached by treatment with 0.25% Trypsin for 10-15 min at 37 °C. Trypsin digestion was halted by the addition of Cell Binding Buffer (DPBS supplemented with 10% Fetal Bovine Serum (FBS) and 1.0% w/v NaN3 (NaN3 added to prevent the internalization of cell surface markers) to a total volume of 13-15 mL. Cells were added to 15 mL falcon tubes and subject to two cycles of centrifugation (300 g, 5 min, room temp.), removing the supernatant after each cycle and diluting with 14 mL of Binding Buffer before the second cycle. Cells were re-suspended in 1.0 mL of Cell Binding Buffer and counted with a BioRad TC20 automated cell counter. Live cell counts were > 95% as indicated by trypan blue staining. The cell concentrations were adjusted to 2.0 x 10 6 cells/mL with additional Binding Buffer. 100 µL aliquots of the cell suspensions were transferred to Eppendorf tubes and placed on ice.</p><!><p>To each 100µL cell aliquot was added 31.3 µL of 0.048 µg/µL Trastuzumab scFv construct (either -GGY tagged or un-tagged and subject to abTYR-mediated O.C. conditions with or without O.G.-488 or abTYR) so that 1.5 µg of scFv was added to each sample. Samples were incubated on ice for 1 hour in the dark. Samples were then subject to 3 cycles of centrifugation (300 g, 3 min), re-suspending cells in 1000 µL of Cell Binding Buffer before each cycle and discarding supernatant after each cycle. After centrifugation, cell pellets were re-suspended in 1000 µL of DPBS with 0.5% paraformaldehyde and kept on ice for 1-3 hours until ready for flow cytometry analysis.</p><!><p>To each 100µL cell aliquot was added 2.0 µL of 1.5 µg/µL un-tagged Trastuzumab scFv. Samples were incubated on ice for 1 hour, protected from light. Samples were then subject to 2 cycles of centrifugation (300 g, 3 min), resuspending cells in 1000 µL of Cell Binding Buffer before each cycle and discarding supernatant after each cycle. † pD was determined from pH meter reading (pH*) by applying the formula 4 : pD = 0.929pH* + 0.42.</p><!><p>Small molecule N-Acetyl-L-Tyrosine and p-Toluidine tyrosinase-mediated oxidative coupling adduct:  Protein L (∆ Domain5) -GGGGSGGY, OC   Gating scheme was applied uniformly to all samples and statistics are tabulated. Fluorescence intensity of single cells was measured using the 488 laser line and FITC filter set.</p>