Genome-scale metabolic model-based engineering of Escherichia coli enhances recombinant single-chain antibody fragment production.

PURPOSE: Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as single-chain variable fragments (scFvs). AntiEpEX-scFv is a small antibody fragment that has received considerable attention for its ability to target the epithelial cell adhesion molecule (EpCAM), a cancer-associated biomarker of solid tumors. Due to its metabolic burden, scFv recombinant expression causes a remarkable decrease in the maximum specific growth rate of the scFv-producing strain. In the present study, a genome-scale metabolic model (GEM)-guided engineering strategy is proposed to identify gene targets for improved antiEpEX-scFv production in E. coli. METHODS: In this study, a genome-scale metabolic model of E. coli (iJO1366) and a metabolic modeling tool (FVSEOF) were employed to find appropriate genes to be amplified in order to improve the strain for incresed production of antiEpEX-scFv. To validate the model predictions, one target gene was overexpressed in the parent strain Escherichia coli BW25113 (DE3). RESULTS: For improving scFv production, we applied the FVSEOF method to identify a number of potential genetic engineering targets. These targets were found to be localized in the glucose uptake system and pentose phosphate pathway. From the predicted targets, the glk gene encoding glucokinase was chosen to be overexpressed in the parent strain Escherichia coli BW25113 (DE3). By overexpressing glk, the growth capacity of the recombinant E. coli strain was recovered. Moreover, the engineered strain with glk overexpression successfully led to increased scFv production. CONCLUSION: The genome-scale metabolic modeling can be considered for the improvement of the production of other recombinant proteins.

Generation and utility of a single-chain fragment variable monoclonal antibody platform against a baculovirus expressed recombinant receptor binding domain of SARS-CoV-2 spike protein.

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

Evaluating the efficacy of immobilized metal affinity chromatography (IMAC) for host cell protein (HCP) removal from anti-HER2 scFv expressed in Escherichia coli.

Host cell proteins (HCPs) are process-related impurities that have influence on product safety and efficacy. HCPs should effectively be removed by chromatographic steps in downstream purification process. In this study, we aimed to evaluate the efficacy of immobilized-metal affinity chromatography (IMAC) for separation of HCPs from anti-HER2 single chain fragment variable (scFv) expressed in E. coli. This study explored how different purification conditions including native, denaturing and hybrid affect HCP level in purified anti-HER2 scFv. Furthermore, the effects of NaCl concentration in wash buffer as well as imidazole concentration in wash and elution buffer on purification yield and HCP level in purified anti-HER2 scFv were evaluated. It was found that increasing imidazole concentration in wash and elution buffers in native conditions reduced the yield of anti-HER2 scFv purification. However, enhancing NaCl concentration in wash buffer in purification under native conditions led to significant increase in the amount of anti-HER2 scFv without any change in protein purity. Herein, none of the IMAC purification methods conducted on soluble cytoplasmic proteins under native conditions could reduce the amount of HCP to acceptable level. HCP content was only lowered to ˂ 10 ppm when inclusion bodies were purified under hybrid conditions. Furthermore, increasing imidazole concentration in wash buffer in purification under hybrid conditions led to significant increase in eluted anti-HER2 scFv concentration, while HCP content was also increased in this condition. Overall, purification under hybrid conditions using wash buffer containing 40 mM imidazole resulted in the highest yield and acceptable level of HCP.

Expression of a Tagless Single-Chain Variable Fragment (scFv) of Anti-TNF-α by a Salt Inducible System and its Purification and Characterization.

BACKGROUND: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases, such as rheumatoid arthritis and Crohn's disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb's auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α. OBJECTIVE: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer. METHODS: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out. RESULTS: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ~ 28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry. CONCLUSION: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Screening of biotin-binding FV-antibodies from autodisplayed FV-library on E. coli outer membrane.

This study aimed to isolate FV-antibodies with biotin-binding activity from a FV-antibody library that was successfully screened on the outer membrane of E. coli. The aims were achieved by (1) preparing a library of FV-antibodies on the outer membrane of E. coli using autodisplay technology, (2) screening the FV-antibodies with biotin-binding activity from the FV-antibody library, and (3) synthesizing peptides (molecular weight of several kDa) from the biotin-binding amino acid sequence of FV-antibodies. An FV-antibody library with a diversity of 1.7 × 105 clones was prepared on the outer membrane of E. coli, using a surface display method called autodisplay technology. For the screening of biotin-binding FV-antibodies, the fluorescence-labeled biotin was introduced into the library, and the target E. coli with biotin-binding activity were screened using flow cytometry. For the screened E. coli clones, the binding affinity (KD) of Fv-antibodies against biotin was calculated and the binding properties of the screened FV-antibody were analyzed through competition assay with a synthetic peptide having the biotin-like activity. From the FRET experiment with the synthetic peptide corresponding to the CDR3 region of the screened Fv-antibody, the biotin-binding activity of the screened FV-antibody was proved to be originated from the CDR3. Finally, the applicability of the biotin-binding domain was demonstrated through the co-expression with a protein called Z-domain with antibody binding activity.

The PAR promoter expression system: Modified lac promoters for controlled recombinant protein production in Escherichia coli.

Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific "promoter activity ratings" (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.

Effective production of single-chain variable fragment (scFv) antibody using recombinant Escherichia coli by DO-stat fed-batch culture.

Dissolved oxygen (DO)-stat fed-batch culture, which allows a high cell density culture of microorganisms under constant DO conditions, was applied to anti-CRP single-chain variable fragment (scFv) production using recombinant Escherichia coli. The DO-stat fed-batch culture was successfully performed under various DO conditions for more than 50 h, resulting in increased scFv production from 0.5 to 0.8 g/L by flask and batch cultures to 2.8-3.0 g/L by the fed-batch culture under the conditions of 5-40% of DO saturation. The formation of inclusion bodies was effectively depressed during DO-stat fed-batch operation; consequently, the solubility of anti-CRP scFv was significantly improved from 36-43% by the flask and batch cultures to 96-98% by the DO-stat fed-batch culture under a wide range of DO conditions. From the kinetic analysis of fed-batch experiments, it was also found that the successful folding of anti-CRP scFv in the cytoplasm occurred when metabolic rates, such as the specific growth rate and specific glucose consumption rate, were relatively low. These results show that the fed-batch culture operated by the DO-stat feeding strategy was effective for the enhanced production of anti-CRP scFv with high solubility.

Expression and purification of a novel single-chain diabody (scDb-hERG1/ß1) from Pichia pastoris transformants.

In the last decades, protein engineering has developed particularly in biotechnology and pharmaceutical field. In particular, the engineered antibody subclass has arisen. The single chain diabody format (scDb), conjugating small size with antigen specificity, offers versatility representing a gold standard for a variety of applications, spacing from research to diagnostics and therapy. Along with such advantages, comes the challenge of optimizing their production, improving expression systems, purification procedures and stability. All such parameters are detrimental for protein production in general and above all for recombinant antibody expression, which has to be fine-tuned, choosing a proper protein-expression host and adjusting expression protocols accordingly. In the present paper, we present data regarding the production and purification of a single chain diabody directed against the macromolecular complex hERG1/ß1 integrin. We focus on the expression of clones deriving from the transformation of Pichia pastoris yeast cells. In particular, we compare two different clones arose from two separate transformation processes, demonstrating that both are suitable for proper protein expression. Moreover, we have set up an expression protocol and compared the yields obtained using two purification machines: Akta Pure and Akta Start, with a positive outcome.

Development of a platform process for the production and purification of single-domain antibodies.

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two-step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

Recombinant Proteins Co-Expressed and Co-Purified in the Presence of Antibody Fragments.

Recombinant antibodies in single-domain format (VHHs) have been recently used for stabilizing antigens during their purification and crystallization. VHHs are also known for their structural stability and a significant part of them share the characteristic of remaining functionally folded also in the absence of the internal disulfide bond. Therefore, they can be expressed as intrabodies in the cell cytoplasm as well as in the bacterial periplasm. This evidence means that, in theory, VHHs can be co-expressed with their antigens independently on the redox constrains. It has also suggested the idea of using co-expression and co-purification of antigen-antibody complexes for maximizing the stabilizing effect of the antibody on its antigen during all the production steps for both cytoplasmic and periplasmic expression strategies.

Expression of a Large Single-Chain 13F6 Antibody with Binding Activity against Ebola Virus-Like Particles in a Plant System.

Pathogenic animal and human viruses present a growing and persistent threat to humans worldwide. Ebola virus (EBOV) causes zoonosis in humans. Here, two structurally different anti-Ebola 13F6 antibodies, recognizing the heavily glycosylated mucin-like domain (MLD) of the glycoprotein (GP), were expressed in transgenic Nicotiana tabacum plants and designed as inexpensive and effective diagnostic antibodies against Ebola virus disease (EVD). The first was anti-EBOV 13F6 full size antibody with heavy chain (HC) and light chain (LC) (monoclonal antibody, mAb 13F6-FULL), while the second was a large single-chain (LSC) antibody (mAb 13F6-LSC). mAb 13F6-LSC was constructed by linking the 13F6 LC variable region (VL) with the HC of mAb 13F6-FULL using a peptide linker and extended to the C-terminus using the endoplasmic reticulum (ER) retention motif KDEL. Agrobacterium-mediated plant transformation was employed to express the antibodies in N. tabacum. PCR, RT-PCR, and immunoblot analyses confirmed the gene insertion, transcription, and protein expression of these antibodies, respectively. The antibodies tagged with the KDEL motif displayed high-mannose type N-glycan structures and efficient binding to EBOV-like particles (VLPs). Thus, various forms of anti-EBOV plant-derived mAbs 13F6-FULL and LSC with efficient binding affinity to EBOV VLP can be produced in the plant system.

Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display.

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.

Construction of genetically modified Lactococcus lactis that produces bioactive anti-interleukin-4 single-chain fragment variable.

Interleukin 4 (IL-4) is a cytokine that induces T-cell differentiation and the production of antibodies from B cells, and plays a crucial role in the allergic response. Therefore, development of a therapeutic approach against IL-4 signaling is expected to prevent or control Th2-related allergic diseases. IL-4 single-chain fragment variable (scFv), which is a recombinant protein consisting of the Fv region of an IL-4 antibody connected to a flexible peptide linker, is expected to be an inhibitor of IL-4 signaling. In this study, recombinant IL-4 scFv was produced by genetically modified lactic acid bacteria (gmLAB); this system is gaining attention as a type of microbial therapeutics. Recombinant gene expression was confirmed with western blotting, and the IL-4 recognition ability of IL-4 scFv produced by gmLAB was examined with an enzyme-linked immunosorbent assay. The macrophage cell line, Raw264.7, and peritoneal macrophages isolated from C57BL/6 mice were employed for an in vitro IL-4 signaling inhibition assay. IL-4 stimulation increased the mRNA expression of arginase-1, a biomarker of IL-4 signaling in macrophages, but arginase-1 expression was suppressed by IL-4 scFv produced by gmLAB, indicating that IL-4 scFv has IL-4 signaling inhibitory activity. gmLAB that produces bioactive IL-4 scFv that was constructed in this study could be an attractive approach for treating allergic disorders.

Anti-tumor effect of single-chain antibody to Reg3a in colorectal cancer.

BACKGROUND: Regenerating protein 3a (Reg3a) is a trophic factor that functions as a stimulus in cell proliferation and neogenesis. Previous studies showed that Reg3a is ectopically upregulated in a majority of colorectal cancers (CRC) and detectable in the serum. METHODS: Single-chain variable fragment targeting Reg3a (scFv-Reg3a) was screened from a phage library. The bioactivity of recombinant Reg3a (rReg3a) and scFv-Reg3a were tested in LoVo and RKO cell lines using MTT, flow cytometry, wound healing and transwell analyses. Whether scFv-Reg3a inhibits tumor growth and enhances 5-fluorouracil (5-FU)-caused cell death were further examined in LoVo cell-transplanted nude BALB/c mice. RESULTS: A scFv-Reg3a from clone C2 was obtained and its binding affinity (KD) to rReg3a was determined to be 4.44 × 10-10. In cultured LoVo and RKO cells, rReg3a promoted but scFv-Reg3a inhibited cell proliferation, survival, migration and invasion. In LoVo cell-xenografted nude mice, administration of rReg3a accelerated tumor growth while scFv-Reg3a suppressed cell proliferation and reinforced 5-FU-induced cell death. CONCLUSION: The newly developed scFv-Reg3a is an anti-cancer agent which is potent to suppress CRC cell proliferation and survival. The use of scFv-Reg3a could enhance the effectiveness of 5-FU-based chemotherapy in the cancerous treatment.

A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs.

Phage-displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full-length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate-limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage-displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage-displayed synthetic single-chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single-chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage-derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell-surface receptors, and scIgGs behave the same as conventional IgGs.

Development of an artificial antibody specific for HLA/peptide complex derived from cancer stem-like cell/cancer-initiating cell antigen DNAJB8.

BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

High efficiency CHO cell display-based antibody maturation.

Previously, we developed a CHO cell display-based antibody maturation procedure in which an antibody (or other protein) gene of interest was induced to mutate by activation-induced cytidine deaminase (AID) and then form a library by simply proliferating the CHO cells in culture. In this study, we further improved the efficiency of this maturation system by reengineering AID, and optimizing the nucleic acid sequence of the target antibody gene and AID gene as well as the protocol for AID gene transfection. These changes have increased both the mutation rate and the number of mutation type of antibody genes by more than 10 fold, and greatly improved the maturation efficiency of antibody/other proteins.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Site-Directed Modification of Yeast-Produced Proteins Using Expressed Protein Ligation.

Expressed protein ligation (EPL), using non-self-cleaving inteins, allows for the site-specific addition of customized chemical moieties to the termini of proteins. In this way, protein activity can be preserved while functionalizing the target protein with a wide range of chemical handles. Here, we describe methods for EPL-based modification of proteins produced by yeast, employing an engineered, non-self-cleaving intein known as 202-08. Methods for EPL modification of both yeast surface displayed and secreted proteins with bioorthogonal chemical groups are described. These methods allow for the site-specific modification of intein-fused proteins produced in yeast.