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A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments

Single-chain variable fragments (scFvs), composed of variable domains of heavy and light chains of an antibody joined by a linker, share antigen binding capacity with their parental antibody. Due to intrinsically low solubility and stability, only two Escherichia coli-produced scFvs have been approved for therapy. Here we report that a 33-residue peptide, termed P17 tag, increases the solubility of multiple scFvs produced in Escherichia coli SHuffle strain by up to 11.6 fold. Hydrophilic sequence, especially charged residues, but not the predicted α-helical secondary structure of P17 tag, contribute to the solubility enhancement. Notably, the P17 tag elevates the thermostability of scFv as efficiently as intra-domain disulfide bonds. Moreover, a P17-tagged scFv targeting hepatitis B virus surface proteins shows over two-fold higher antigen-binding affinity and virus-neutralizing activity than the untagged version. These data strongly suggest a type I intramolecular chaperone-like activity of the P17 tag. Hence, the P17 tag could benefit the research, production, and application of scFv.

tissue penetration capability and are more rapidly cleared than mAbs 3,7 , making them particularly suitable for applications such as diagnostic in vivo imaging and therapeutic targeting of tumors and intracellular infectious agents, including viruses. Moreover, in contrast to costly mAb production in mammalian cells, Ab fragments can be principally be produced in prokaryotes such as Escherichia coli (E. coli) or Brevibacillus choshinensis at a much lower cost and in higher yield 8 . Despite these small-size-related benefits, Fc-less Ab fragments, especially Fv segments, are less stable than mAbs and show an increased tendency to unfold and aggregate under thermal stress. This is mainly attributed to the lack of inter-and intra-chain disulfide bonds and additional non-covalent interactions provided by the Fc domain 8,9 . Aggregates of Ab fragments can induce undesired immunogenicity and toxicity, resulting in compromised pharmacokinetic and therapeutic properties 2 . Strategies to improve the stability of Fv segments include reinforcing the interface between the variable domains of H and L chains (VH and VL) via covalent linkages in some formats, such as dsFv or scFv where two Fvs are covalently linked by a disulfide bond 10 or a flexible peptide 11 , and sc-dsFv where two Fvs are linked by a disulfide bond plus a peptide 12 . Short (5-10 aa) peptide linkers to connect VH with VL promote intermolecular VH-VL interactions (called domain-swapping) by sterically disfavoring intramolecular VH-VL interactions, giving rise to dimers (also termed diabodies) 13 . Additional strategies have been developed to improve solubility and stability of bacterially expressed scFvs, including directed evolution, rational design, co-expression of chaperones, and fusion with solubilityenhancing tags 8 . However, despite these efforts, until 2020 only eight Ab fragments, two scFvs of them produced in E.coli, have been approved for clinical use by the US Food and Drug Administration 8 .
Intradomain disulfide bonding between two β-sheets of a VH or VL domain is often important to stabilize scFv. Mutations of the involved cysteines increase the propensity for aggregation 14,15 . The cytoplasmic compartment of common E. coli strains not only lacks components to catalyze the formation of disulfide bonds but instead possesses active glutathione reductase (gor) and thioredoxin reductase (trxB) pathways to reduce disulfide bonds 16 . Hence most cytoplasmically expressed scFvs are obtained as non-functional insoluble aggregates. While the periplasmic space, in contrast, provides an oxidative environment and contains thiol-disulfide oxidoreductases such as DsbA and DsbB 17 , recombinant expression yields are mostly very low. Recently, soluble expression of some scFvs has been achieved in an engineered E. coli strain (termed SHuffle) which features a more oxidative and disulfidepromoting cytoplasmic environment due to inactivation of the gor and trxB pathways and the constitutive expression of the disulfide-bond isomerase DsbC [18][19][20][21] . Also, other E. coli strains co-expressing a redoxactive enzyme (e.g., Erv1p, DsbB or VKOR) plus a disulfide-bond isomerase like DsbC or PDI have become available [22][23][24][25] . However, some scFvs still exhibit low solubility in these engineered E. coli strains 25 and the low stability of scFvs remains problematic 8 .
As reported, folding of select scFvs is predominantly mediated by intrinsically strong VH-VL interactions, independent of intradomain disulfide bond 9,26 . Their at least partly soluble cytoplasmic expression in E. coli is thus not unexpected. Fusion with some protein tags such as maltose-binding protein (MBP), glutathione-S-transferase (GST), N utilization substance A (NusA) and thioredoxin (Trx) can further improve the solubility [27][28][29] . However, for many scFvs with weak VH-VL interactions, this is unlikely the case 28 . In addition, it remains tricky and laborious to refold insoluble scFvs into active forms using detergents and additives, although fusion to MBP may improve expression and refolding yields 28 . However, it is often mandatory to remove bulky tags like MBP to preserve the scFv's antigen-binding and pharmacokinetic properties 30 . In turn, tag removal often destabilizes the scFv and induces non-functional aggregate formation 29,30 .
The P17 protein from the tail of T7 phage can target phage particles yet also other cargos including small molecules, proteins, siRNA, DNA polyplexes, and liposomes to hepatocytes in vitro and in vivo 31,32 . This is predominantly mediated by the association of a 33-amino acid (aa) P17 protein segment (named P17 peptide or P17 tag hereafter) with cell-surface low density lipoprotein receptor-related protein (LRP) 33 . We previously generated an scFv of mAb G12 18,34 , which targets the surface protein of hepatitis B virus (HBV), a virus with pronounced liver tropism. To improve hepatocyte targeting and uptake of G12-scFv 18 , here we attach the P17 tag to its C-terminus. When expressed in the E. coli SHuffle strain, the G12-scFv-P17 fusion protein displays much higher solubility than the untagged G12-scFv. Furthermore, the P17 tag also increases the solubility of three other scFvs in SHuffle cells, as revealed by the semiquantitative solubility assay. The current study aims to identify the sequence and structure determinants within the P17 peptide that are responsible for its solubility and stabilityenhancing impact on scFvs, and to clarify the underlying mechanisms.

Results
P17 tag enhanced solubility of four different scFvs in E. coli SHuffle T7 strain Plasmid pET28a-His-G12-scFv-HA expresses a G12-scFv protein with a His tag on the N terminus and a HA tag at the C-terminus 18 . Into this plasmid, the P17 tag coding sequence was inserted downstream of the HA tag sequence, with the resulting plasmid pET28a-His-G12-scFv-HA-P17 encoding the same protein but now with the additional P17 tag at the C-terminus. E. coli SHuffle T7 cells transformed with either of the two plasmids were cultured in Luria-Bertani (LB) media supplemented with 0.5 mM IPTG to express recombinant G12-scFv-HA and G12-scFv-HA-P17 proteins, and subsequently were suspended in 10 ml lysis buffer per gram (wet weight) of bacteria. After cell rupture by sonification, 10 μl of each lysate containing total bacterial protein was mixed with 40 μl 2× SDS-PAGE loading buffer as the source of total scFv. After centrifugation, 10 μl of the clear lysate supernatant containing soluble bacterial protein was mixed with 40 μl 2× SDS-PAGE loading buffer as the source of soluble scFv. To determine the solubility of scFv defined as the percentage ratio of soluble to total scFv, both fractions of scFvs were semi-quantitated by western blotting using a mAb against the common His tag followed by a horseradish peroxidase-conjugated secondary antibody plus a chemiluminescent substrate, and subsequent densitometry of the blotting signals recorded by a chargecoupled device camera (Fig. 1a, upper panel). To avoid signal saturation, both fractions of scFvs were diluted serially by 1/5, 1/10, 1/20, 1/40, 1/80, and 1/160 before loading. As shown in Fig. 1a (middle panel), western blotting signals of both fractions of scFvs correlated logarithmically with the dilution. However, at high dilutions ranging from 1/ 160 to 1/20, signal intensities displayed an approximately linear correlation with the dilution (Fig. 1a, bottom panel). Therefore, for quantitation, soluble scFv was diluted in the range from 1/20 to 1/80, while total scFv was diluted by 1/160 due to the higher amount. ScFv solubility was assessed after normalizing the dilution of soluble scFv at 1/160. As shown in the left panel of Fig. 1b, the solubility of G12-scFv-HA and G12-scFv-HA-P17 proteins was 14.9% and 45.0%, respectively, indicating the P17 tag increased G12-scFv solubility.
In line with our previous report 18 , the G12-scFv-HA protein was virtually insoluble when expressed in BL21 (DE3) Star cells, which harbor active gor and trxB pathways preventing intradomain disulfide bond formation (Fig. 1b, right panel). Also, the G12-scFv-HA-P17 protein remained insoluble in this strain (Fig. 1b, right panel), indicating that the solubility-enhancing effect of the P17 tag required an oxidative cytoplasmic environment.
To check whether the P17 tag could enhance the solubility of other scFvs, recombinant pET28-derived plasmids were constructed to express three additional scFvs: VRC01-scFv targeting gp120 of HIV-1 35 , ADRI-scFv targeting the small surface protein of HBV 36 , and MA18/7-scFv targeting preS1 domain of the large HBV surface protein 37 , either with or without the P17 tag at their C termini. As shown in Fig. 1c, d, the solubility of these scFvs expressed in SHuffle T7 strain showed some variations, but fusion with the P17 tag increased in all cases solubility from 2.4 to 6.5-fold. The solubility-promoting effect of the P17 tag was most remarkable for MA18/7-scFv, with its untagged version having the lowest solubility (Fig. 1d).
In the scFv constructs described above, a HA tag was located between scFv and P17 tag to facilitate immunological detection. To check whether the HA tag contributed to the solubility-enhancing effect of the P17 tag, we constructed an analogous plasmid, pET28a-His-MA18/7-scFv-P17, which lacks the HA coding sequence. In direct comparison with MA18/7-scFv-HA, the solubility of this new construct, MA18/7-scFv-P17, was even more elevated (by 9.5-fold) (Fig. 1c, d), indicating that the additional presence of the HA tag decreased solubility enhancement by the P17 tag.   Both N-and C-terminal fusions of the P17 tag efficiently increased scFv solubility The impact of a tag on the solubility of a fusion protein can be affected by the tag's location at the N or C-terminus of a protein of interest 38 . Therefore, we compared the solubility of MA18/7-scFv with the P17 tag directly linked to either end (Fig. 2a). As shown in Fig. 2b-d, the P17 tag significantly improved solubility at both N-and C-positions by 11.6-and 10.2-fold, respectively. In the additional presence of a HA tag at the Cterminus, the P17 tag increased solubility by 3.5-fold when at the N terminus and 5.9-fold when at the very C-terminus (Fig. 2d). Therefore, direct attachment of the P17 tag to either terminus of the scFv increased solubility efficiently, whereas an additional HA tag at the scFv C-terminus reduced the P17-mediated solubility enhancement.
A C-terminal P17 tag enhanced G12-scFv solubility more efficiently than N-or C-terminally fused MBP in SHuffle T7 cells Multiple fusion tags, including MBP, have been applied to improve the solubility of scFvs in E. coli [27][28][29] . Indeed, N-terminal but not C-terminal MBP fusion increased the solubility of G12-scFv by 1.4-fold in SHuffle T7 cells (Fig. 3c) as revealed by the solubility assay ( Fig. 3a, b). C-terminal P17 tag fusion increased the solubility of G12-scFv by 1.7-fold, in line with a higher soluble yield of G12-scFv-P17 than G12-scFv-HA and MBP-G12-scFv ( Supplementary Fig. S3b). Hence in SHuffle T7 cells, the P17 tag increased the solubility of G12-scFv more efficiently than MBP. Glutathione-S-transferase (GST) is another protein tag that is commonly used to improve the solubility of passenger proteins in E. coli 39 , often as N-terminal fusion 40 . Its effect on the solubility of G12-scFv was therefore also tested. However, the GST-G12-scFv-HA protein was even much less soluble in SHuffle T7 cells than the N-terminally untagged version ( Fig. 3a-c).
Adding two or three copies of P17 tags did not further improve scFv solubility The solubility-enhancing ability of the P17 tag may be endowed by recruiting other molecules such as chaperones or perhaps tRNAs [41][42][43][44] to promote protein folding. If so, adding two or three copies of the P17 tag might further elevate the solubility of a test scFv. To test this hypothesis, two or three copies of the tag were fused to the C-terminus of MA18/7-scFv. The results showed that a single P17 tag increased solubility by 8.3-fold, whereas two and three copies enhanced solubility by only 6.6-and 3.8-fold, respectively (Fig. 4).
A predicted α-helix in the P17 tag is not required for solubility enhancement Structural features of a protein tag can be important for solubility enhancement 45,46 . The free P17 peptide did not exhibit any characteristic α-helical structure when analyzed by circular dichroism spectroscopy 31 , but an earlier study of the entire P17 protein had noted a substantial α-helix propensity in the rod-region harboring the P17 tag sequence 47 . In the absence of direct structural information, we applied up-to-date secondary structure prediction algorithms, JPred4 48 and PEP2D 49 , to the P17 peptide (see Supplementary Fig. S1a for the JPred4 results), and the three-dimensional (3D) structure prediction program AlphaFold2 50 to the entire MA18/7-scFv-HA-P17 protein ( Supplementary  Fig. S1b). Either program predicted a single region within the P17 tag to have some α-helix propensity, namely residues 22-29 (RDEAKRFK, Fig. 5a) near the C-terminus, although the AlphaFold2 predicted local Distance Difference Test (plDDT) score in this region was below 70 (Supplementary Fig. S1b), indicating an at most moderate confidence in the predicted α-helix. However, the α-helix might still form in the scFv context or be induced by interaction with bacterial components, c Solubility of His-MA18/7-scFv-HA and its derivatives were determined by three independent experiments. Data are presented as mean values ± SD. The statistical significance of differences between two experimental groups was assessed through one-way analysis of variance (ANOVA). d Fold change of solubility of P17-tagged derivatives of His-MA18/7-scFv-HA to untagged version.
notably the abundant tRNA, as in the recently described "chaperna" mechanism where RNA is crucial for chaperoning [41][42][43][44] . Different from the RNA interaction domain used there, we could not detect a stable association of the P17 tag with RNA ( Supplementary Fig. S2), disfavoring such a mechanism. To further experimentally assess whether the putative α-helix, if formed, was important for solubility enhancement by the P17 tag, we replaced the alanine in the center of the putative helix, A25, by a helix-breaking proline residue 51 (Fig. 5a, variant A25P); JPred4, as well as alphafold2, predicted indeed a loss of the α-helix (Supplementary Fig. S1a, b, variant A25P). When attached to the C-terminus of MA18/7-scFv-HA, both the wild-type (wt) and A25P mutant P17 tag increased protein solubility by around fourfold (Fig.  5b-d). Furthermore, a variant lacking the entire sequence forming the putative α-helix (variant P17-C15del), was at most slightly less efficient than the wt P17 tag to increase the solubility of MA18/7-scFv (see below, Fig. 6a-d). Therefore, the putative α-helix structure, if formed at all, was dispensable for solubility enhancement.
Both N-and C-proximal sequences in the P17 tag contribute to scFv solubility enhancement The 33-aa P17 tag is highly hydrophilic due to the presence of eight basic aa (R, K), five acidic aa (D, E), and twelve polar aa (e.g., N, Q, S, T), all of which may contribute to the tag's solubility-enhancing property.
The solubility-enhancing ability of the P17 tag correlated with the number of charged residues Polyanionic and polycationic protein tags have been reported to improve the solubility of some recombinant proteins 46 . As 15-aa P17-N18del retained wt-like solubility-enhancing ability, it was used for subsequent point-mutation analysis. Because two anionic residues (D23, E24) and two cationic residues (K26, R27) are clustered in the central region of P17-N18del ( Fig. 7a), we investigated their potential roles by site-directed mutants (P17-N18del-KR, P17-N18del-DE, and P17-N18del-DEKR) with D23/E24 and/or K26/R27 replaced alone or in combination by alanine (Fig. 7a) were fused to the C-terminus of MA18/ 7-scFv. In the context of the P17 tag, all mutants were predicted to maintain a wt-like α-helix propensity between residues R22-K29 (Supplementary Fig. S1a, there named P17 DE23,24AA and KR26,27AA). Hence, these variants would also test for the importance of positive and negative charges in the putative α-helix, even if its formation was unlikely (see above). All these point mutants still increased solubility of MA18/7-scFv significantly, though to variable extents ( Fig. 7b-d).

The P17 tag increases scFv stability
Protein tags can enhance solubility by assisting in the correct folding of the fusion partners 45,46 . This reduces exposure of hydrophobic regions to solvent and inhibit formation of aggregates. Protein stability decreases with increasing temperature and can be reflected by the melting temperature (Tm) at which the concentrations of folded and unfolded proteins are equal 52 . Generally, a higher Tm indicates higher stability and vice versa 52 . As reported previously, the Tm of a test protein can be determined by a thermal shift assay 52,53 . In this assay, fluorescent dye (SYPRO Orange) binds to hydrophobic regions exposed during heat denaturation, yielding more 570-nm-wavelength quanta 53 . As determined by this assay, treatment of G12-scFv-HA with 5 mM DTT to reduce disulfide bonds led to~3°C decrease of Tm (Fig. 8a), in line with the presence of such disulfides and their importance of intradomain disulfide bonds to maintain scFv stability in previous studies 14,15 . Next, to check the effect of P17 tag on scFv stability, recombinant G12-scFv-HA and G12-scFv-HA-P17 proteins were firstly purified by IMAC (Supplementary Fig. S3a). Quantitation of both proteins by SDS-PAGE and Coomassie-Blue staining using BSA as standard revealed soluble yields of 3 mg and 8 mg per liter culture (Supplementary Fig. S3b), corroborating the solubility enhancement by the P17 tag. Five μM solutions of both proteins were mixed with equal concentrations of SYPRO Orange and subjected to the thermal shift assay. As shown in Fig. 8b, G12-scFv-HA-P17 displayed a 2.3°C higher Tm than G12-scFv-HA, indicating that the P17 tag stabilized scFv by inhibiting exposure of hydrophobic regions. As a control, incubation in trans with in vitro synthesized P17 peptide failed to increase the Tm of G12-scFv-HA even in 10-fold excess (Fig. 8c), indicating that the P17 tag stabilized scFv only as part of the protein and did so by increasing resistance against unfolding.
P17-tagged G12-scFv has higher antigen binding affinity and specificity and HBV infection-neutralizing activity than the untagged protein Due to its increased stability, the scFv-P17 fusion protein may possess stronger antigen-binding affinity than unfused scFv, unless the tag would sterically hinder antigen binding. To compare the binding affinity of G12-scFv-HA and G12-scFv-HA-P17 to recombinant HBV small surface protein produced in CHO cells, the equilibrium dissociation constants (Kd) were determined by biolayer interferometry. A 2.2-fold lower Kd (0.79 × 10 −10 M versus 1.77 × 10 −10 M, Fig. 9a) for G12-scFv-HA-P17 than for G12-scFv-HA was obtained. These results were further corroborated by enzyme-linked immunosorbent assay (ELISA), whereby the pooled sera of chronic hepatitis B patients and healthy donors, confirmed to be positive and negative for hepatitis B surface antigen (HBsAg) by a commercial ELISA kit (Fig. 9b), were reacted with the microtiter wells coated with 5 μg/ml solutions of G12-scFv-HA and G12-scFv-HA-P17, respectively. ELISA A450nm value of G12-scFv-HA-P17 reacting with HBsAg positive sera was significantly (P < 0.0001), approximately twofold higher than G12-scFv-HA (Fig. 9b). These data collectively indicate stronger antigen binding affinity of G12-scFv-HA-P17 than G12-scFv-HA, and also suggest that the P17 tag did not interfere with scFv-antigen binding. Generally, stronger antigen binding affinity of scFv suggests higher specificity. Indeed, ELISA A450nm value of G12-scFv-HA-P17 incubating with HBsAg negative sera was significantly (P < 0.05) lower than G12-scFv-HA, thus indicating the higher specificity (Fig. 9b). Importantly, both G12-scFv-HA and G12-scFv-HA-P17 proteins efficiently inhibited HBV infection of a susceptible cell line as judged by the dose-dependent suppression of HBV RNAs and secreted hepatitis B e antigen (Fig. 9c). However, G12-scFv-HA-P17 protein displayed higher neutralizing activity than G12-scFv-HA, as indicated by of 50% inhibitory concentrations at 2.91 nM versus 6.74 nM (Fig. 9c, right panel).
P17 tag did not lead to oligomerization of G12-scFv Some protein tags like GST mediate oligomerization of fusion partners by spontaneous self-assembly into dimer or higher oligomers 45,54 . Recombinant full-length P17 protein assembles into trimer, as natively present in T7 phage tail fibers 47 . In contrast, the 33-aa P17 peptide existed as a monomer in solution as determined by circular dichroism and analytical ultracentrifugation 31 . To assess the potential impact of the P17 tag on G12-scFv quaternary structure, recombinant G12-scFv-HA and G12-scFv-HA-P17 proteins were analyzed by size-exclusion chromatography (SEC). As shown in Fig. 10a (left panel), the Superdex 200 Increase 10/300 column separated protein markers ranging from 0.137 kDa to 670 kDa very well, giving the linear calibration curve and equation of average distribution constants (K av ) of each protein marker and the logarithm of the respective molecular weight (Log(MW), Fig. 10a, right panel). Under identical experimental conditions, both the G12-scFv-HA and the G12-scFv-HA-P17 protein displayed two peaks (Fig. 10a, left panel), representing monomeric and dimeric forms of each protein based on the calibration curve and equation. To confirm and its truncated mutants with various deletions of C-or N-terminal residues. Basic, acidic, and polar residues are shown in blue, red, and green, respectively. The putative α-helix from P17 residues 22-29 is denoted with a cylinder. b Representative western blot to determine the solubility of MA18/7-scFv-HA protein and its derivatives fused to P17-WT or the C-terminally truncated mutants P17-C8del and P17-C15del. c Semiquantitative determination of the solubility of MA18/7-scFv-HA protein and its derivatives from b by three independent experiments. d Fold change of solubility of MA18/7-scFv derivatives from b compared to untagged MA18/7-scFv-HA. e Representative western blot to determine the solubility of MA18/7-scFv-HA protein and its derivatives fused to P17-WT or the N-terminally truncated mutants P17-N8del, P17-N14del, and P17-N18del. f Semiquantitative determination of the solubility of MA18/7-scFv-HA protein and its derivatives from e by three independent experiments. g Fold change of solubility of MA18/7-scFv derivatives from e compared to untagged MA18/7-scFv-HA. T total protein, S soluble protein. Data are presented as mean values ± SD. The statistical significance of differences between the two experimental groups was assessed by one-way ANOVA. this interpretation, both protein preparations were cross-linked using 3% (v/v) formaldehyde, and the products were analyzed by SDS-PAGE and subsequent western blotting using the anti-His-tag mAb (Fig. 10b). This revealed dimers as the major higher-order products. The ratios of dimer to monomer were essentially the same for G12-scFv-HA and G12-scFv-HA-P17, both by the intensity of the western blot signals and the areas of the SEC elution peaks (Fig. 10a, b). Taken together, the P17 tag neither resulted in oligomerization of G12-scFv nor did it alter the equilibrium between monomeric and dimeric forms, rather in line with a type I than an assembly-mediating type II intramolecular chaperone mechanism 55 .

Discussion
As one format of antibody fragments, scFv are applied in the fields of basic research, diagnostics, and therapeutics. Amongst different expression hosts 56,57 , E. coli offers rapid growth at low cost 8 . However, scFv are often expressed as insoluble aggregates because common E. coli strains with their reducing cytoplasmic environment fail to   support formation of intramolecular disulfide bond 16 . Strategies to improve scFv solubility in E. coli include expression in the oxidative periplasmic space, reduced culture temperature, co-expression of chaperone(s), using engineered E. coli variants providing a more oxidizing cytoplasm, and fusion of solubility-promoting tags to the scFv 2,8 . Here, we characterized the 33-aa P17 peptide tag, and further shortened variants, as a small-size fusion tag which markedly increases the solubility, soluble yield, and stability of scFvs in E. coli SHuffle cells.

Solubility-enhancing effect of P17 tag on multiple scFvs
Analogous to other protein tags, solubility enhancement of scFvs by the P17 tag depends on both the expression host and properties of the specific scFv 45,58 . Firstly, in line with intradomain disulfide-bond formation promoting scFv solubility in E. coli, G12-scFv was partly soluble in the SHuffle strain but not in the BL21(DE3) strain with its reducing cytoplasm (Fig. 1b). Only in SHuffle cells did the P17 tag further increase scFv solubility (Fig. 1b, d). Secondly, while the P17 tag did not improve solubility of a truncated duck hepatitis B virus polymerase, notorious for poor solubility in E. coli 59 ( Supplementary Fig. S4), the tag enhanced solubility of four different scFvs (Fig. 1d). Part of Ab diversity is due to many subgroups of heavy and light chains. Heavy chains are encoded by a single gene cluster (IGH), light chains by the κ cluster (IGK) and the λ cluster (IGL) 60 . Sequence analysis of the four scFvs using online International ImMunoGeneTics Information System (http://www.imgt.org/) revealed that their variable regions are encoded by different subgroups of heavy (IGHV) and light chain variable gene clusters (IGKV, and IGLV). Accordingly, the variable regions of MA18/7-scFv are encoded by IGHV3 and IGKV6; those of G12-scFv by IGHV4 and IGKV3; those of VRC01-scFv by IGHV1 and IGKV1; and those of ADRI-scFv by IGHV3 and IGLV3. Although these scFvs vary considerably in sequence and their intrinsic solubility properties in E. coli, attaching the P17 tag improved the solubility of all by at least 2.4-fold and up to 11.6-fold ( Figs. 1 and 2). Hence, the P17 tag can improve the solubility of multiple scFvs in the SHuffle strain.

Properties of the P17 tag and implications
Their small sizes of around 30 kDa convey to scFvs efficient tissue penetration and renal glomerular filtration 3 . Oligomerization and the corresponding increase in size would substantially dampen these properties. However, the P17 peptide as such exists as monomer in solution and also as a tag it neither induced oligomerization of scFv nor did it affect the intrinsic scFv monomer-dimer equilibrium (Fig. 10). With about 4 kDa in size the P17 tag is much smaller than most other currently used protein tags. Bulkier tags impair size-related pharmacokinetic scFv properties and also increase the metabolic burden of the expression hosts. Furthermore, bulky tags such as MBP may sterically interfere with the scFv-antigen interaction 61 since MBP-G12-scFv fusion protein displayed significantly lower binding affinity to recombinant HBV small surface protein than untagged G12-scFv-HA ( Supplementary  Fig. S5). Hence larger tags often need to be proteolytically removed 45 , which can destabilize scFvs 29,30 . In our study, the 33-aa P17 tag and its truncated variants, especially P17-N18del mutant comprising only 15 aa (Fig. 6g), improved scFv solubility regardless of terminal fusion positions (Fig. 6). The P17 tag displayed stronger solubility-enhancing effects on scFv than C-terminally fused MBP or N-terminally fused GST (Fig. 3). Most remarkably, the P17 tag also improved thermostability, antigen binding affinity and specificity of scFv (Figs. 8 and 9). Therefore, the P17 tag removal is not mandatory for in vitro use. However, if desired, especially for avoiding immunogenicity in in vivo applications, the P17 tag can be efficiently cleaved off by enterokinase, and possibly other cleavage site/protease combinations ( Supplementary Fig. S3c) after the introduction of an enterokinase digestion site (DDDDK). Importantly, solubility of the tag-less scFv protein was not compromised, as shown by virtually identical band intensities of equal amounts of untreated vs. enterokinase-treated protein after centrifugation at 20,000×g for 30 min (Supplementary Fig. S3c). These data also suggest that the P17 tag facilitates the adoption of a stably folded scFv conformation but is not required to maintain it. For therapeutic and diagnostic purposes, scFvs and diabodies have to maintain antigen-binding activity without aggregating at 37°C for hours to days after injection, requiring high intrinsic stability 8,9 . The P17 tag not only promoted expression solubility in E. coli SHuffle strain but also increased thermostability and antigen binding affinity of G12-scFv. In this way, the P17 tag could benefit long-term storage stability of scFvs, as practically supported by the much fewer insoluble aggregates observed after 1.5 years of cryo-preservation of P17-tagged scFvs. Hence to our knowledge, P17 tag is the first reported peptide tag to increase the thermostability of scFvs.
As reported 33 , P17 peptide targeted multiple cargos including proteins, siRNA, and DNA-liposome complex to hepatocytes by interaction with the receptor LRP on cell surface, leading to endocytosis of cargos by hepatocytes. Indeed, P17-tagged G12-scFv was taken up by primary mouse hepatocytes more efficiently than untagged G2-scFv, as observed by immunofluorescence confocal microscopy (Supplementary Fig. S6). In this regard, besides increasing solubility and production of scFv, P17 tag can specifically augment scFv's effect in hepatocytes by increasing intracellular concentration. In our previous study, G12-scFv targeting hepatitis B surface antigen could be endocytosed by hepatocytes and potently inhibited secretion of HBV virions 18 . Hence a stronger inhibition by P17-tagged G12-scFv than untagged version can be envisaged due to stronger antigen-binding affinity as well as more efficient uptake.

Mechanisms by which P17 tag increases the solubility of scFv in SHuffle strain and scFv stability
Many current protein tags and peptide tags, with molecular weights of 4.5-55 kDa and 0.15-1.6 kDa, respectively, have been developed for enhancing the solubility of proteins of interest in E. coli 46 . Protein tags increase protein solubility by multiple mechanisms including recruitment of chaperones or functioning as intramolecular chaperones or folding nuclei to facilitate protein folding, transformation of the fusion proteins into micelle-like conformations, and enhancement of intraand intermolecular electrostatic repulsive forces 45,46 , whereas peptide tags composed of less than 15 repetitive cationic or anionic residues do so predominantly by increasing electrostatic repulsive forces 46 .
Proteins are least soluble at the pH value equal to their isoelectric point (pI) where they do not bear net charges. Polyionic peptide tags or some protein tags can increase net charges of fusion proteins by rendering pIs more different from physiological pH 46 . This not only promotes interaction with solvent molecules but also increases the time for proper folding via stronger intra-and intermolecular repulsive electrostatic forces within a fusion protein. Such a mechanism is unlikely to apply to the P17 tag because P17-N18del-D23A/E24A, though with more net positive charges, displayed weaker solubility-enhancing ability than P17-N18del instead (Fig. 7d). At physiological pH, the P17 tag, VRC01-scFv, and G12-scFv with predicted pIs of 9.82, 8.84, and 8.35 have positive charges, but MA18/7-scFv and ADRI-scFv with pIs of 5.52 and 5.53 have negative charges (Fig. 1d). Disregarding charge accessibility, the P17 tag increases net positive charges for VRC01-scFv, and G12-scFv, but reduces overall net charges of MA18/7-scFv and ADRI-scFv. Nonetheless, the P17 tag had a weaker solubility-enhancing effect on VRC01-scFv and G12-scFv than on MA18/7-scFv and ADRI-scFv (Fig. 1d), arguing against the supercharging model 46 . Forcing P17tagged scFv into micelle-like structures is also unlikely since SEC only revealed monomeric and dimeric forms of G12-scFv-HA-P17 (Fig. 10a). The same holds for chaperone recruitment by the P17 tag because two or three copies of P17 tags did not increase but rather weaken the solubility enhancement of a single tag copy (Fig. 4). Though it is not excluded that P17 tag residues 22 to 29 adopt an α-helical secondary structure in P17-tagged scFvs, such an α-helix is dispensable for solubility enhancement, as evidenced by point mutation and deletion mutation analysis (Figs. 5 and 6d). We also found no positive evidence for an interaction of the P17 tag with RNA, which could contribute to chaperoning [41][42][43][44] .
Higher thermostability, as well as stronger antigen affinity and virus neutralization activity of P17-tagged versus untagged G12-scFv-HA, suggest that the P17 tag promoted correct folding of scFv via an intramolecular chaperone-like activity. Two types of intramolecular chaperones are currently defined 55 . Type I intramolecular chaperones assist tertiary structure formation at an early stage of protein folding by acting as the folding nucleus or by preventing misfolding and aggregation, while type II intramolecular chaperones are not directly involved in protein folding but play key roles in the formation of quaternary structure 55 . P17 tag, monomeric on its own, did not alter the quaternary conformation of scFv (Fig. 10). Moreover, excess P17 peptide added in trans post purification did not increase thermostability of G12-scFv (Fig. 8b). All these data suggested that P17 tag functions as type I intramolecular chaperone. During folding of the scFv-P17 fusion protein, the P17 tag may interact with hydrophobic regions of folding intermediates of the scFv but also with solvent molecules through the many polar residues. This will promote protein correct folding and inhibit formation of aggregates. Indeed, reducing the number of charged residues impaired the solubility-enhancing ability of the P17 tag (Fig. 7d), similar to non-ATP hydrolyzing holdase chaperones and α-crystallin-derived peptide mini-chaperones [62][63][64][65] .
Besides solubility, stability is the other challenge for practical applications of scFv. The overall stability of two-domain scFvs including the intrinsic stability of VH and VL domains, and the stability of the hydrophobic VH-VL interface relate closely to the specific sequences of the complementary determining regions and the intrinsic stability of subdomains such as β barrels 9 . Intradomain disulfide bonds connecting two β sheets per scFv monomer are also critical for intrinsic domain stability of many scFvs, as one disulfide bond contributes 4-6 kcal/mol of free energy to protein folding 66 . This was corroborated by an~3°C Tm decrease of G12-scFv upon DTT treatment (Fig. 8a). Notably, the P17 tag enhanced the Tm of G12-scFv by 2.3°C (Fig. 8b), thus increasing overall stability of scFv as efficiently as intradomain disulfide bonds. Hence most likely, the scFv stability increase by the P17 tag is as well due to type I intramolecular chaperone activity. Thereby the P17 tag may promote protein folding by increasing the unfolding energy barrier and by stabilizing the hydrophobic VH-VL interface, eventually reducing promiscuous domainswapping to oligomerize monomer and dimer into soluble or insoluble aggregates 9,67 .

Ethical statement
The sera of CHB patients were obtained from Drs. Demin Yu and Xinxin Zhang from Ruijin Hospital of Shanghai JiaoTong University School of Medicine. The research use of these sera has been approved by the Ruijin Hospital Ethics Committee (EC approval No. 2016-124) and got informed consent from the donors.
The plasmids constructed in this study will be freely available for research use from the corresponding authors within 1 month of the request being made.
Protein expression, bacteria lysis, and protein purification E. coli SHuffle T7 (New England Biolabs) or BL21 (DE3) Star (Thermo-Fisher Scientific) strains transformed by pET28a vector-derived scFv expressing plasmids were cultured in LB media containing 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C for 16 h to induce expression of scFv proteins. Bacteria collected by centrifugation at 8,000 g for 5 min were suspended in 10 ml lysis buffer (20 mM Tris-HCl, pH 7.5, 0.5% Triton X-100, 150 mM NaCl, and 10 mM MgCl 2 ) supplemented with 5 μg/ml DNaseI, 50 μg/ml RNaseA, 1 mg/ml lysozyme, and 1× protease inhibitor cocktail solution (Roche) per gram, and lysed on ice by 12 repeats of ultrasonic disruption with 99 cycles of sonication at 80 Watt for 1 sec and break for 1 s. Ten μl bacterial lysate representing the total bacterial protein fraction was mixed with 40 μl 2× SDS-PAGE loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 12% glycerol, 4 mM DTT, and 0.02% bromophenol blue) for western blotting analysis of total scFv protein. After centrifugation of bacterial lysate at 12,000×g at 4°C for 20 min, 10 μl clear supernatants representing soluble fraction were mixed with 40 μl 2× SDS-PAGE loading buffer for western blotting analysis of soluble scFv protein. Soluble scFv protein in the supernatant was purified by immobilized metal ion affinity chromatography (IMAC) with an Ni-nitrilotriacetic acid (Ni-NTA) superflow column (Qiagen), followed by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare). ScFvs were finally dialyzed against PBS and concentrated by Amicon centrifugal ultrafiltration devices (Millipore) with a molecular cutoff of 3 kDa.

SEC
Two hundred μl protein markers (catalog No: 69385, Sigma) or test scFvs diluted in PBS were loaded onto a Superdex 200 Increase 10/300 column using ÄKTA Purifier system (GE Healthcare). Proteins eluted by PBS at a flow rate of 0.5 ml/min were monitored via ultraviolet absorbance at 280-nm wavelength. According to the user manual of the column, the average distribution constant (K av ) of test protein was determined according to the equation K av = (V e − V 0 )/(V c − V 0 ) using the parameters: elution volume (V e ) of test protein, void volume (V 0 ) and bed volume (V c ) of the column. The calibration curve of SEC was plotted as K av of protein markers versus logarithms of their molecular weights using the linear fitting program of Origin 8.0 software.

Biolayer interferometry
Biolayer interferometry to measure Kd reflecting binding affinity of G12-scFv-HA or G12-scFv-HA-P17 to hepatitis B surface antigen (HBsAg) was performed as previously described 18 . Amine-reactive sensors were coated with 5 µg/ml CHO cell-expressed S protein in sodium acetate buffer at pH 5.0 as recommended by the manufacturer, then set to bind various concentrations of G12-scFv-HA or G12-scFv-HA-P17 protein at 37°C for 300 s, and dissociate in PBS for another 600 s in an Octet Red96 System (Pall ForteBio). Interference signals produced by the association or dissociation of molecules were recorded and Kd was obtained with the Octet QK software package.

Thermal shift assay
Thermal shift assays were performed as previously described 53 with minor modifications. SYPRO Orange dye was firstly mixed with 5 μM final concentration of scFv according to user manual (ThermoFisher Scientific). An optical 96-well reaction plate (Applied Biosystems) loaded with 20 μl reaction mixture per well was mounted onto a realtime PCR instrument (QuantStudio 6 Flex, Applied Biosystems). The mixture was heated up by 0.05°C/s from 30 to 90°C, and the fluorescence signal in 570-nm wavelength yielded by the dye was recorded concomitantly. Melting temperature (Tm) for each scFv was determined by fitting fluorescence signal and temperature to the Boltzmann equation using Protein Thermal Shift software v1.3 (ThermoFisher Scientific).

Production of HBV, neutralization assay, and northern blotting
These assays were performed as previously reported 69 . Briefly, HBV particles secreted by HepAD38 cells were precipitated with 7.5% PEG8,000 solution and quantitated by real-timer quantitative PCR using HBV-specific primers. HBV at a nominal multiplicity of infection of 100 vge/cell was incubated with various concentrations of scFv at room temperature for 30 min, and then was used to inoculate 1.5 × 10 5 HepG2-NTCP cells per well grown on a collagen-coated 24-well plate in the presence of 4% PEG8,000. After 16 h, cells were extensively washed by 500 μl PBS per well for five times and cultured with DMEM containing 2.5% DMSO until harvest. At day 6 post HBV infection, hepatitis B e antigen (HBeAg) secreted by HBV-infected cells in the culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA) following the user manual. Total RNA was extracted from HBVinfected cells using TRIzol reagents (ThermoFisher) according to the manufacturer's protocol. HBV RNA was detected by northern blotting using HBV-specific 32 P-labeled DNA probe 69 . Densitometry of hybridization signals was done using MultiGauge V2.2 (Fujifilm).
The HepG2-NTCP cell line was previously established 18 and here authenticated by morphology, western blotting of NTCP protein, or PCR with NTCP-specific primers. HepAD38 cell line was provided by Dr. Christoph Seeger (Fox Chase Cancer Center, Philadelphia, USA) and authenticated by morphology and PCR with HBV-specific primers.

Immunofluorescence microscopy
The assay was conducted as previously reported 18 . Primary mouse hepatocytes grown on collagen-coated coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. After blocking with 3% bovine serum albumin (fraction V, Sigma) for 10 min, cells were incubated with the respective primary antibodies followed by Alexa Fluor 488 or Cy3 conjugated secondary antibodies. Nuclear DNA was stained with 4', 6-diamidino-2-phenylindole (Sigma). Cells were visualized by a laser confocal scanning microscope (Leica TCS SP8). Image processing was done by Las AF Lite 2.6 software (Leica).

Statistics and reproducibility
Results acquired by three independent experiments were presented as average values ± standard deviations. Statistical significance of differences between two experimental groups was assessed one-way analysis of variance as implemented in Origin 8.0 software. Fifty percent inhibitory concentrations of scFvs were also calculated by Origin 8.0 software.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Source data are provided with this paper.