Production Of Antibody Fragments Research Articles

Optimizing recombinant antibody fragment production: A comparison of artificial intelligence and statistical modeling.

Maximizing the recombinant protein yield necessitates optimizing the production medium. This can be done using a variety of methods, including the conventional "one-factor-at-a-time" approach and more recent statistical and mathematical methods such as artificial neural network (ANN), genetic algorithm, etc. Every approach has advantages and disadvantages of its own, yet even when a technique has flaws, it is nevertheless used to get the best results. Here, one categorical variable and four numerical parameters, including post-induction time, inducer concentration, post-induction temperature, and pre-induction cell density, were optimized using the 232 experimental assays of the central composite design. The direct and indirect effects of factors on the yield of anti-epithelial cell adhesion molecule extracellular domain fragment antibody were examined using statistical methods. The analysis of variance results indicate that the response surface methodology (RSM) model is effective in predicting the amount of produced single-chain fragment variable (p-value = 0.0001 and R2 = 0.905). For ANN modeling, the evaluation using normalized root mean square error (NRMSE) and R2 values shows a good fit (R2 = 0.942) and accurate predictions (NRMSE = 0.145). The analysis of error parameters and R2 of a dataset, which contained 30 data points randomly selected from the complete dataset, showed that the ANN model had a higher R2 value (0.968) compared to the RSM model (0.932). Furthermore, the ANN model demonstrated stronger predictive ability with a lower NRMSE (0.048vs. 0.064). Induction at the cell density of 0.7 and an isopropyl β-D-1-thiogalactopyranoside concentration of 0.6mM for 32 h at 30°C in BW25113 was the ideal culture condition leading to the protein yield of 259.51mg/L. Under the optimum conditions, the output values predicted by the ANN model (259.83mg/L) were more in line with the experimental data (259.51mg/L) than the RSM (276.13mg/L) expected value. This outcome demonstrated that the ANN model outperforms the RSM in terms of prediction accuracy.

RNA-seq reveals multifaceted gene expression response to Fab production in Escherichia coli fed-batch processes with particular focus on ribosome stalling

BackgroundEscherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations.ResultsProduction of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg2+, indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising.ConclusionsThrough characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production.

Protein engineering strategies contribute efficient production of therapeutic antibody fragments

Advances in production methods make antibody fragments a valuable tool in therapeutic applications.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

Scale‐related process heterogeneities change properties of high‐cell‐density fermentation broths demonstrated with Escherichia coli B and K‐12 strains

AbstractBACKGROUNDThe scale‐up of Escherichia coli bioprocesses for production of recombinant proteins in the biopharmaceutical industry can affect process performance due to gradients occurring at production scale. The main objective of this study was to investigate the effect of scale‐related process heterogeneities on properties of high‐cell‐density fermentation broths. Two industrially relevant Escherichia coli strains, BL21(DE3) and HMS174(DE3), were used for production of antibody fragments (Fab). To generate these heterogeneities, we used a scale‐down device for cell cultivation, which mimics conditions of production scale (>1000 L) in a 20 L laboratory scale. This setup helps to evaluate the impact of process scale‐up on the resulting fermentation broth properties relevant for primary recovery.RESULTSWe found differences in broth viscosity between the two strains, but also between the standard laboratory‐scale and the scale‐down cultivations used. An increase in cell size was measured in scale‐down cultivations with three orthogonal methods, using scanning electron microscopy, dynamic light scattering and a dispersion analyzer. Strong hints for an altered agglomeration tendency of the cells after scale‐down cultivations were found by measurement of settling velocity and viscoelasticity of the fermentation broth.CONCLUSIONSIn this work, industrially relevant fermentation broths of Escherichia coli cells producing Fab were characterized and represent a relevant basis for rational design of downstream processing operations. The findings show that scale‐related process heterogeneities can alter the phenotype of Escherichia coli cells and thus alter fermentation broth properties relevant for primary recovery. Considering scale‐up effects in upstream processing reflected in downstream processing will help to optimize the entire bioproduction chain in the future. © 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Production and applications of fluorobody from redox-engineered Escherichia coli

Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E. coli hosts tested, an engineered E. coli B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2–8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets.Key points• E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody)• Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal• This platform can be used to produce fluorobodies for numerous purposesGraphical abstract

Expression in Pichia pastoris of human antibody fragments that neutralize venoms of Mexican scorpions

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.

Production of endoglin-specific heavy chain antibody fragments (VHHs) microarrays for whole-cell SPR imaging

Cell-based Surface Plasmon Resonance imaging (SPRi) with anti-endoglin VHH antibodies microarray on a gold-covered biochip was used to characterize the binding to endoglin expressed on the surface membrane of SC cells. SPRi showed the specific capture of SC cells by the immobilized anti-endoglin VHHs, but not of THP-1 cells that do not express endoglin on their plasmatic membrane. The SPRi reflectivity shifts with SC cells bound by anti-endoglin VHHs ranged from 3.59 to 5.54% (average 4.66 ± 1.18%) and with THP-1 cells from 2.23 to 3.05% (average 2.62 ± 1.32% (p < 0.001)) and the SC cell densities with anti-endoglin VHHs ranged from 417 to 747.7, (average 548 cells/2.28 × 105 μm2 ± 132). Anti-endoglin VHHs bind epitopes located in the extracellular domains of endoglin with different avidity as suggested by the differences in SC cell densities. The cell-based SPRi of anti-endoglin VHH microarrays can be used both in studies aimed at a better understanding of endoglin function in living cells and in VHH applications as nanoprobes.

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

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. 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). 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. The genome-scale metabolic modeling can be considered for the improvement of the production of other recombinant proteins.

Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris.

The interest in the use of monoclonal antibodies as therapeutic molecules has raised in the recent years. Due to their high affinity and specificity towards other biological molecules, antibodies are being widely used to treat a broad range of human diseases such as cancer, rheumatism, and cardiovascular diseases. Currently, the production of IgG-like antibodies is mainly obtained from stable or transient mammalian expression systems that allow proper folding and posttranslational modifications. Despite the technological advances of the last decade, the use of these systems still has a rather high production cost and long processing times. For these reasons, researchers are increasingly interested in alternative antibody production methods as well as alternative antibody formats. Bacterial systems, such as Escherichia coli, are extensively being used for recombinant protein production because their easy manipulation and cheap costs. However, the presence of lipopolysaccharides (LPS) traces in the already fractionated recombinant protein makes these systems not good candidates for the preparation of therapeutic molecules. Yeast systems, such as Pichia pastoris, present the convenient easy manipulation of microbial systems but show some key advantages of eukaryotic expression systems, like improved folding machinery and absence of LPS. They are especially suitable for the production of antibody fragments, which do not need human-like glycosylation, avoiding the high costs of mammalian systems. Here, the protocol for the expression and purification of a single-chain antibody fragment (scFv) in P. pastoris is provided, in deep detail for lab manipulation and briefly for a 5L-bioreactor production.

A novel strategy for efficient expression of an antibody fragment in Escherichia coli: ranibizumab as a case study

AbstractBACKGROUNDComplex biotherapeutics such as non‐glycosylated antibody fragments can be easily manufactured in Escherichia coli due to advancements in recombinant DNA technology. Ranibizumab is one such example of a complex, non‐glycosylated protein used to treat age‐related macular degeneration and macular oedema. Ranibizumab, a humanized monoclonal antibody fragment, is often expressed as inclusion bodies using the E. coli expression system. In this study, we propose a novel strategy for achieving efficient expression of antibody fragments in E. coli BL21 (DE3). Here, the whole antibody fragment (heavy chain + light chain of ranibizumab) has been engineered and cloned in a pET series vector under single promoter belonging to a prokaryotic host along with an extra ribosome binding site to allow equal expression of the light and heavy chains.RESULTSWe demonstrate heterologous expression of ranibizumab with a protein concentration of 0.4 ± 0.019 mg mL–1, confirmed using reversed‐phase high‐performance liquid chromatography with the double copy clone. The reduced and refolded antibody fragment have been analytically characterized using liquid chromatography–mass spectrometry, where masses of the in‐house clone were in correspondence with marketed product. Native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR) analyses were performed to confirm the formation of purified and active recombinant product. Binding kinetics to the target analyte vascular endothelial growth factor of refolded in‐house product was found to be similar to the marketed product as per SPR (12.7 nmol L−1 vs. 10.4 nmol L−1).CONCLUSIONCloning and process optimization have been performed to enhance expression yield. The proposed strategy offers an efficient approach for enhanced production of antibody fragments in microbial hosts. © 2021 Society of Chemical Industry (SCI).

Implementation of a Design of Experiments to Improve Periplasmic Yield of Functional ScFv Antibodies in a Phage Display Platform.

Purpose: Production of functional recombinant antibody fragments in the periplasm of E. coli is a prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effective and lab-scale production of antibody fragments demands the optimization of culture conditions. Methods: The culture conditions such as temperature, optical density (OD600) at induction, induction time, and IPTG concentration were investigated to optimize the functional expression of a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effects of different culture media and osmolyte supplements on the expression yield of scFv were examined. Results: The developed 2FI regression model indicated the significant linear effect of the incubation temperature, the induction time, and the induction OD600 on the expression yield of functional scFv. Besides, the statistical analysis indicated that two significant interactions of the temperature/induction time and the temperature/induction OD600 significantly interplay to increase the yield. Further optimization showed that the expression level of functional scFv was the most optimal when the cultivation was undertaken either in the TB medium or in the presence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine. Conclusion: In the present study, for the first time, we successfully implemented DoE to comprehensively optimize the culture conditions for the expression of scFv molecules in a phage antibody display setting, where scFv molecules can be isolated from a tailor-made phage antibody library known as "Human Single Fold scFv Library I."

The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments.

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise "uncrystallizable" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.

Solubility, Stability, and Avidity of Recombinant Antibody Fragments Expressed in Microorganisms.

Solubility of recombinant proteins (i.e., the extent of soluble versus insoluble expression in heterogeneous hosts) is the first checkpoint criterion for determining recombinant protein quality. However, even soluble proteins often fail to represent functional activity because of the involvement of non-functional, misfolded, soluble aggregates, which compromise recombinant protein quality. Therefore, screening of solubility and folding competence is crucial for improving the quality of recombinant proteins, especially for therapeutic applications. The issue is often highlighted especially in bacterial recombinant hosts, since bacterial cytoplasm does not provide an optimal environment for the folding of target proteins of mammalian origin. Antibody fragments, such as single-chain variable fragment (scFv), single-chain antibody (scAb), and fragment antigen binding (Fab), have been utilized for numerous applications such as diagnostics, research reagents, or therapeutics. Antibody fragments can be efficiently expressed in microorganisms so that they offer several advantages for diagnostic applications such as low cost and high yield. However, scFv and scAb fragments have generally lower stability to thermal stress than full-length antibodies, necessitating a judicious combination of designer antibodies, and bacterial hosts harnessed with robust chaperone function. In this review, we discuss efforts on not only the production of antibodies or antibody fragments in microorganisms but also scFv stabilization via (i) directed evolution of variants with increased stability using display systems, (ii) stabilization of the interface between variable regions of heavy (VH) and light (VL) chains through the introduction of a non-native covalent bond between the two chains, (iii) rational engineering of VH-VL pair, based on the structure, and (iv) computational approaches. We also review recent advances in stability design, increase in avidity by multimerization, and maintaining the functional competence of chimeric proteins prompted by various types of chaperones.

Advances in Production of Monoclonal Antibody and Antibody Fragments in Engineered Prokaryotes and Yeast

Advances in Production of Monoclonal Antibody and Antibody Fragments in Engineered Prokaryotes and Yeast

Understanding unfolding and refolding of the antibody fragment (Fab). I. In-vitro study

In-vitro protein refolding is a major rate-limiting step in the large scale production of antibody fragments expressed using a microbial source like E. coli. This investigation is focused on understanding the in-vitro unfolding and refolding of the multi-domain protein involving inter-domain disulfide linkage, like antibody fragment (Fab). Solubilization behavior of the inclusion bodies and unfolding events of Fab fragment (Biosimilar rHu Ranibizumab) were studied using nano-differential scanning fluorimetry (nano-DSF). Fab unfolding behavior was studied by fitting experimental data with the two-state and three-state thermodynamic model. Based on the Fab unfolding understanding, a two-stage design of experiment (DoE) strategy was used for the optimization of the in-vitro refolding condition of a Fab fragment. Refolding yield of 56.03 ± 1.15 % was achieved using the optimized oxidative refolding conditions maintained by appropriate dilution factor and redox reagent ratio. Refolding kinetics of the rHu Ranibizumab was analyzed using a three-parameter kinetic model showing rate constant k1:7.05e-6 l/mg.min, k2:0.57 l/mg.min, and k3:310.19 l/mg.min. Based on observed refolding kinetics, it was concluded that the Fab refolding follows a three-state mechanism with the refolding intermediate/(s) formation from light and heavy chain of the Fab fragment as an overall rate-limiting step. The method described here is a useful tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.

Enhancement of extracellular bispecific anti-MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition.

Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti‐MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti‐MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti‐MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region

BackgroundRecombinant proteins are often engineered with an N-terminal signal peptide, which facilitates their secretion to the oxidising environment of the periplasm (gram-negative bacteria) or the culture supernatant (gram-positive bacteria). A commonly encountered problem is that the signal peptide influences the synthesis and secretion of the recombinant protein in an unpredictable manner. A molecular understanding of this phenomenon is highly sought after, as it could lead to improved methods for producing recombinant proteins in bacterial cell factories.ResultsHerein we demonstrate that signal peptides contribute to an unpredictable translation initiation region. A directed evolution approach that selects a new translation initiation region, whilst leaving the amino acid sequence of the signal peptide unchanged, can increase production levels of secreted recombinant proteins. The approach can increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coli.ConclusionsThe study demonstrates that signal peptide performance is coupled to the efficiency of the translation initiation region.

Effective Strategies to Overcome the Insolubility of Recombinant ScFv Antibody against EpCAM Extracellular Domain in E. coli

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein overexpressed in epithelial-derived neoplasms and therefore is a highly interesting target for antibody therapy in a wide range of carcinomas. Single chain variable fragment (ScFv) antibodies, generated by the association of the variable heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker, retain the binding properties of classical antibodies. Due to its characteristics, Escherichia coli (E. coli) has inspired a great deal of interest for production of antibody fragments with high concentrations. Here, ScFv against EpCAM extracellular domain (EpEX) was expressed in E. coli BL21™(DE3) strain. The effect of different expression conditions on the total protein level was also investigated. Moreover, an attempt was made to overcome the problem of insolubility of the recombinant protein with alterations of expression condition like inducer concentration and temperature as well as addition of the solubility-enhancing agents. Our results showed that the maximum total protein expression was attained 7 h after induction at 37 °C with 0.5 mM IPTG (663.53 ± 7.33 mg/l). Moreover, the expressed antiEpEX-ScFv protein was 39.8% or 29.1% soluble in the presence of 50% glycerol, and Tween20 plus 50% glycerol respectively. Although the solubility of recombinant protein was significantly increased from 39.8% at 37 °C to 43.7% at 16 °C, the maximum level of soluble recombinant protein was attained at 37 °C. Consequently, we report a strategy combining different culture conditions and the solubility-enhancing additives such as glycerol for improving protein solubility.