Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

Expression of an scFv against HER2 derived from Trastuzumab in SHuffle at 30 °C resulted in enhanced solubility and higher expression level compared to its expression in BL21(DE3) at 37 °C.

“Recently, it has been reported that the expression of an scFv against HER2 derived from Trastuzumab (drug bank number DB00072) in SHuffle at 30 °C resulted in enhanced solubility and a higher expression level of the molecule as compared to its expression in BL21 (DE3) at 37 °C [109,110,111,112].”

• cites [109] Ahmadzadeh, 2020, Anti-HER2 scFv expression in Escherichia coli SHuffle((R))T7 express cells: Effects on solubility and biological activity, Mol. Biotechnol. sourceNot checked — no full text of the source
• cites [110] Akbari, 2014, Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli, J. Ind. Microbiol. Biotechnol. sourceDisputed

  The Results and discussion (page unavailable) identify the scFv as derived from pertuzumab, not trastuzumab. The paper reports expression in BL21(DE3), with optimal production at 37 °C and the highest soluble-to-insoluble ratio at 25 °C; it does not report the claimed SHuffle-versus-BL21 comparison. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [111] Verdoliva, 1995, Topological mimicry of cross-reacting enantiomeric peptide antigens, J. Biol. Chem. sourceCould not verify

  The paper’s Results discuss antibody cross-recognition of P15 peptide variants, not recombinant scFv expression. Searches for trastuzumab, HER2, SHuffle, and BL21(DE3) found no passage addressing the claimed solubility or expression comparison. The available passages have no page numbers. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [112] Russo, 2017, Ultra-performance liquid chromatography/multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions, Rapid Commun. Mass Spectrom. sourceNot checked — no full text of the source

The final production yield of anti-HER2 scFv was 147 mg/L under optimal expression conditions (24 h after induction with 0.05 mM IPTG at 30 °C, LB medium, SHuffle strain).

“The final production yield of the anti-HER2 scFv was 147 mg/L, under optimal expression conditions (24 h after induction with 0.05 mM IPTG at 30 °C, LB medium, SHuffle strain).”

• cites [109] Ahmadzadeh, 2020, Anti-HER2 scFv expression in Escherichia coli SHuffle((R))T7 express cells: Effects on solubility and biological activity, Mol. Biotechnol. sourceNot checked — no full text of the source
• cites [110] Akbari, 2014, Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli, J. Ind. Microbiol. Biotechnol. sourceDisputed

  The Results and discussion section states that optimal anti-HER2 scFv production occurred with 1 mM IPTG at 37 °C, contrary to the claimed 0.05 mM at 30 °C. The Bacterial strains, plasmid, and reagents section identifies BL21(DE3), not SHuffle, as the expression host and describes 2 hours of induction, not 24. The cited passages do not establish the claimed 147 mg/L yield; page numbers are unavailable in our copy. (LLM, conf=0.89)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [111] Verdoliva, 1995, Topological mimicry of cross-reacting enantiomeric peptide antigens, J. Biol. Chem. sourceCould not verify

  The Materials and Methods and Results passages describe synthetic peptide antigens and anti-MAP-P15 antibodies, not anti-HER2 scFv production. Searches for the 147 mg/L yield, SHuffle strain, IPTG induction, and the stated expression conditions found no passage addressing the claim. No page numbers were provided for these passages. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [112] Russo, 2017, Ultra-performance liquid chromatography/multiple reaction monitoring mass spectrometry quantification of trastuzumab in human serum by selective monitoring of a specific peptide marker from the antibody complementarity-determining regions, Rapid Commun. Mass Spectrom. sourceNot checked — no full text of the source

High-affinity binding of VHH and V-NAR domains is likely supported by an increased length of the majority of CDR3 loops (3-28 amino acids) compared to VH domains.

“Moreover, the high-affinity binding of VHH and V-NAR domains is likely supported by an increased length of the majority of the CDR3 loops (3–28 amino acids) compared to that of the VH domains [13].”

• cites [13] Mitchell, 2018, Analysis of nanobody paratopes reveals greater diversity than classical antibodies, Protein Eng. Des. Sel. sourcePartly supported

  The Introduction says VHH nanobodies achieve high binding affinities and have H3 loops around 15% longer than classical antibody H3 loops; the Conclusion links those loops to antigen contacts and interaction specificity. The paper does not establish that greater loop length supports high affinity, and the cited passages do not address V-NAR domains, a majority of loops, or the 3–28-amino-acid range (page numbers unavailable). (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Camelid VHH and shark V-NAR domains are used as templates to generate highly efficient and stable nanobodies.

“Given their high-affinity, solubility and stability also in absence of the partner VL domain, the camelid VH domains (VHH) [4] and the shark VH domains called V-NAR (New Antigen Receptor) [5], are currently used as templates to generate highly efficient and stable new antibody fragments collectively termed nanobodies [6,7,8].”

• cites [4] Harmsen, 2007, Properties, production, and applications of camelid single-domain antibody fragments, Appl. Microbiol Biotechnol. sourceSupported
• cites [5] English, 2020, Ancient species offers contemporary therapeutics: An update on shark VNAR single domain antibody sequences, phage libraries and potential clinical applications, Antib. Ther. sourceSupported
• cites [6] Jovcevska, 2020, The therapeutic potential of nanobodies, BioDrugs sourcePartly supported

  The Introduction (“Nanobodies, the Smaller Variant of Antibodies”; page not provided) identifies camelid VHH domains as nanobodies and describes their affinity, solubility, and stability. It does not establish that shark V-NAR domains are used as templates for nanobodies; the shark-related material found is a reference title, not a finding of this paper. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [7] 2020, Recombinant expression of nanobodies and nanobody-derived immunoreagents, Protein Expr. Purif. sourcePartly supported

  The Introduction (page unavailable) identifies camelid VHH domains as nanobodies and describes their binding affinity, structural stability, and engineering into reagents. It does not support the claim that shark V-NAR domains are also used as templates for nanobodies; searches for V-NAR and shark yielded no relevant passage. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [8] Cossins, 2007, Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L, Protein Expr. Purif. sourceNot checked — no full text of the source

Fab and scFv are the most used and widespread antibody-like formats.

“Despite that several alternative antibody-like formats derived from different Ig-like domain combinations are continuously developed and proposed [1] such as single-domain antibody fragments (dAbs), the antigen-binding fragments (Fab) and single-chain variable fragments (scFv) are those most used and widespread.”

• cites [1] Bates, A., and Power, A.C. (2019). David vs. goliath: The structure, function, and clinical prospects of antibody fragments. Antibodies, 8. sourcePartly supported

  Section 2.2 reports that Fabs account for approximately 49% of antibody fragments entering clinical trials, supporting their prominence. Section 2.1 gives examples of scFvs in clinical development, but the paper does not establish that Fab and scFv are together the most used and widespread formats across all applications. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Karyolaimos and coworkers obtained a yield of around 0.2 g/L of a functional scFv fragment in the periplasm using the OmpA signal peptide and 100 μM rhamnose as inductor.

“Karyolaimos and coworkers have obtained a yield of around 0.2 g/L of a functional scFv fragment in the periplasm using the OmpA signal peptide and 100 μM rhamnose as inductor [68].”

• cites [68] Karyolaimos, 2019, Enhancing recombinant protein yields in the E. coli periplasm by combining signal. Peptide and production rate screening, Front. Microbiol. sourceSupported

Use of fusion partners such as GST, MBP, SUMO and Trx to target proteins, especially scFv fragments, results in solubility-enhancing properties and increased yields of soluble and active products.

“Several reports have also suggested that the use of fusion partners such as GST [118], maltose-binding protein (MBP) [119,120], small ubiquitin-related modifier (SUMO) [87,121] and thioredoxin (Trx) [122] to the target proteins, especially to scFv fragments, results in solubility-enhancing properties and in increased yields of soluble and active products.”

• cites [118] Sina, 2015, Effects of environmental factors on soluble expression of a humanized Anti-TNF-alpha scFv antibody in Escherichia coli, Adv. Pharm. Bull. sourceCould not verify

  The citation [122] is attached specifically to thioredoxin (Trx), not to the other fusion partners. The Introduction describes a GST-fused scFv, and the Discussion and Conclusion attribute changes in its soluble expression to cultivation conditions; the passages found for thioredoxin and Trx do not address whether a Trx fusion increases soluble, active product yields. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [119] Bach, 2001, Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies, J. Mol. Biol. sourceNot checked — no full text of the source
• cites [120] Yang, 2018, Screening of a ScFv antibody with high. affinity for application in human IFN-gamma immunoassay, Front. Microbiol. sourcePartly supported

  The Results (Soluble Expression of ScFv) show that MBP fusion improved scFv solubility, supporting the MBP portion to which this citation is attached. The Discussion says MBP fusion alone did not improve binding activity; a linker was needed for the highest activity. The paper does not establish increased yields of active products from MBP fusion alone. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [87] Rezaie, 2017, Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase((R)) cultivation mode, J. Appl. Microbiol. sourceNot checked — no full text of the source
• cites [121] Ye, 2008, High.-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli, Appl. Microbiol. Biotechnol. sourceSupported

CyDisCo has been exploited as an efficient route for production of scFv and Fab fragments derived from known antibodies of different classes and organisms in the cytoplasm of the KEIO K12 E. coli strain in shake flasks.

“The CyDisCo has been exploited as an efficient route for the production of scFv and Fab fragments derived from known antibodies of different classes and from different organisms (human, mouse and humanized) in the cytoplasm of the KEIO (collection parental K12) E. coli strain in shake flasks [127].”

• cites [127] Baba, 2006, Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection, Mol. Syst. Biol. sourcePartly supported

  The Results and discussion section (“Keio collection mutants”; page unavailable) identifies BW25113 as the E. coli K-12 strain used to construct the Keio collection, supporting the strain identification. It does not report CyDisCo production of scFv or Fab fragments, the antibody origins, or shake-flask production, so it does not support the sentence’s main production claim. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

High local concentrations of nascent heterologous protein together with insufficient amount of folding-promoting chaperons may lead to partially folded or misfolded protein intermediates that give rise to formation of insoluble protein aggregates known as inclusion bodies in both cytoplasm and periplasm.

“High local concentrations of the nascent heterologous protein together with an insufficient amount of folding-promoting chaperons may lead to partially folded or misfolded protein intermediates that give rise to the formation, in both the cytoplasm and periplasm, of insoluble protein aggregates known as inclusion bodies, IBs [77].”

• cites [77] Upadhyay, A.K., Murmu, A., Singh, A., and Panda, A.K. (2012). Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli. PLoS ONE, 7. sourceSupported

With such high-expression yields in flasks, production of antibody fragments would not require scale-up to bioreactors during drug development.

“With such high-expression yields in the flasks, the production of antibody fragments would not require a scale-up to bioreactors during the drug development process [129].”

• cites [129] Lagasse, 2017, Recent advances in (therapeutic protein) drug development, F1000 Res. sourceCould not verify

  The Protein engineering section discusses the complexity of therapeutic-protein manufacturing, but does not address antibody-fragment yields in flasks or whether those yields eliminate bioreactor scale-up. Searches for those terms and for flask production without scale-up found no passage supporting the claim; page numbers were not available in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

In the presence of cumate, the pNEW vector increases production yield of recombinant proteins by two to three-fold compared to pET-based IPTG-inducible systems.

“In the presence of cumate, the pNEW vector is able to increase the production yield of recombinant proteins by two to three-fold compared to pET-based IPTG-inducible systems.”

• cites [72] Choi, 2010, Novel, versatile, and tightly regulated expression system for Escherichia coli strains, Appl. Environ. Microbiol. sourceSupported

Kulmala and coworkers demonstrated that harmonized versions of Fab fragments rather than the classic over-optimized one increase expression from negligible levels to 10 mg/L.

“Recently, Kulmala and coworkers have demonstrated that the “harmonized versions” of Fab fragments rather than the classic “over-optimized” one increases the expression from negligible levels to 10 mg/L [60].”

• cites [60] Kulmala, 2017, Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli, Sci. Rep. sourceSupported

The anti-HER2 scFv yield with CyDisCo was 50.3% higher than the yield obtained without CyDisCo using only EnPresso B (167 mg/L).

“which is 50.3% higher than the yield obtained without CyDisCo using only EnPresso B (167 mg/L)”

• cites [54] Gaciarz, 2016, Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli, Microb. Cell Fact. sourceSupported

At the transcriptional level, codon harmonization has largely improved expression of antibody fragments.

“At the transcriptional level, the concept of “codon harmonization”, a sophisticated version of the codon usage optimization, has largely improved the expression of antibodies fragments [60,103].”

• cites [60] Kulmala, 2017, Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli, Sci. Rep. sourcePartly supported

  The Abstract and Discussion report that codon harmonization increased expression of a human Fab fragment in E. coli from negligible levels to 10 mg/l. The claim generalizes that result to antibody fragments and places the effect at the transcriptional level; the Discussion instead describes a suggested mechanism involving translation kinetics and protein folding. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [103] Mignon, 2018, Codon harmonization—Going beyond the speed limit for protein expression, FEBS Lett. sourcePartly supported

  In “Expression of CAI profile optimized sequences” (page unavailable), the paper reports that codon-optimized sequences markedly increased protein expression of scFv D1.3, an antibody fragment. It does not establish an improvement at the transcriptional level: the reported outcome is protein abundance, and the paper frames codon usage chiefly in terms of translation. Its direct result also concerns one antibody fragment, not antibody fragments generally. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Production of an anti-HER2 scFv with CyDisCo was 251 mg/L using EnPresso B media.

“The production of an anti-HER2 scFv with CyDisCo was 251 mg/L using EnPresso B media [54]”

• cites [54] Gaciarz, 2016, Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli, Microb. Cell Fact. sourceSupported

Correct protein folding in the E. coli periplasm is completed and checked by PPIases such as SurA, FkpA, PpiA, PpiD, Skp and DegP.

“The process leading to correct protein folding in the E. coli periplasm is further completed and checked by the activity of PPIases such as SurA, FkpA, PpiA, PpiD, Skp, and DegP (see Figure 1) [137,138,139,140].”

• cites [137] Saul, 2004, Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity, J. Mol. Biol. sourceNot checked — no full text of the source
• cites [138] Schafer, 1999, Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins, J. Biol. Chem. sourcePartly supported

  The Abstract identifies PpiA, FkpA, SurA and PpiD as periplasmic PPIases. But the Abstract and Discussion identify Skp as a molecular chaperone involved in early folding, while the Abstract identifies DegP as a protease; they do not support calling either a PPIase or saying this group completes and checks folding. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [139] Sklar, 2007, Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli, Genes Dev. sourcePartly supported

  The paper’s discussion of chaperone roles (page unavailable) supports a role for SurA, Skp, and DegP in outer-membrane protein folding and assembly, and identifies SurA as a member of the PPIase family. But it describes Skp and DegP as general chaperones, not PPIases, and says the chaperones responsible for folding periplasmic proteins remain an open question. It does not establish the claimed role for PpiA or PpiD. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [140] Schlapschy, 2011, Periplasmic chaperones used to enhance functional secretion of proteins in E. coli, Methods Mol. Biol. sourceNot checked — no full text of the source

Two new peptides selected from signal peptide libraries improved the periplasmic production of active 13R4 by approximately 40% compared to expression obtained using wild type pelB.

“According to the signal related to β-lactamase activities, two new peptides were selected from the libraries as improving the periplasmic production of active 13R4 by ~40% compared to the expression obtained using the wild type pelB [98].”

• cites [98] Williams, 2018, Development of a generic beta-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli, Sci. Rep. sourceSupported

The OmpA-leader sequence was selected as the best in plasmid-free expression systems for the production of a functional Fab fragment.

“Recently, the OmpA-leader sequence (MKKTAIAIAVALAGFATVAQA) was also selected as the best in plasmid-free expression systems (GI) for the production of a functional Fab fragment [74,104].”

• cites [74] Hausjell, J., Kutscha, R., Gesson, J.D., Reinisch, D., and Spadiut, O. (2020). The effects of lactose induction on a plasmid-free, E. coli T7 expression system. Bioengineering, 7. sourcePartly supported

  Materials and Methods §2.1 (page unavailable) identifies the stated OmpA-leader sequence on both chains of a genome-integrated Fab construct. The Introduction identifies FabZ as the antigen-binding fragment used. The paper does not report comparing leader sequences or selecting OmpA as the best. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [104] Fink, 2019, Microbioreactor cultivations of fab-producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct fab expression effects, Biotechnol. J. sourcePartly supported

  Section 3.1 and Table 1 describe genome-integrated, plasmid-free systems using OmpA to produce Fab fragments. But Section 3.3.1 says Fab yields with DsbA were not clearly different from those with OmpA and that the authors could not generally conclude which translocation route was favorable; it does not support selecting OmpA as the best leader sequence. Page numbers were not available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

The 22 amino acids long PelB signal sequence from Erwinia carotovora is the most frequently used for transportation of Fabs and scFvs.

“The 22 amino acids long PelB (pectate lyase B) signal sequence from Erwinia carotovora [157] is the most frequently used for transportation of the Fabs [88,96] and scFvs [30,101,158,159].”

• cites [157] Lei, 1987, Characterization of the Erwinia carotovora pelB gene and its product pectate lyase, J. Bacteriol. sourceSupported
• cites [88] Rodriguez, 2017, Efficient antibody assembly in E. coli periplasm by disulfide bond. Folding factor Co-expression and culture optimization, Appl. Biochem. Biotechnol. sourceCould not verify

  Citation [157] is attached to the description of PelB as a 22-amino-acid signal sequence from Erwinia carotovora. The Materials and Methods section (page unavailable) says the authors used pelB leader peptides for a Fab expression vector, but our copy does not state the sequence length or organism of origin. Searches for those details found no supporting passage. (LLM, conf=0.73)

  Experimental — accuracy not yet measured
• cites [96] Kumar, D., Batra, J., Komives, C., and Rathore, A.S. (2019). QbD based media development for the production of fab fragments in E. coli. Bioengineering, 6. sourceSupported
• cites [30] Dewi, 2016, Construction and periplasmic expression of the Anti-EGFRvIII ScFv antibody gene in Escherichia coli, Sci. Pharm. sourceSupported
• cites [101] Yousefi, 2018, Soluble expression of humanized Anti-CD20 single chain antibody in Escherichia coli by cytoplasmic chaperones co-expression, Avicenna J. Med. Biotechnol.Not checked — no full text of the source
• cites [158] Demeu, L.M.K., Soares, R.J., Miranda, J.S., Pacheco-Lugo, L.A., Oliveira, K.G., Cortez Plaza, C.A., Billiald, P., Ferreira de Moura, J., Yoshida, N., and Alvarenga, L.M. (2019). Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion. PLoS ONE, 14. sourceSupported
• cites [159] Na, 2019, Extracellular production of functional single-chain variable fragment against aflatoxin B1 using Escherichia coli, Lett. Appl. Microbiol. sourceNot checked — no full text of the source

The pelB signal sequence resulted largely superior over the DsbA signal peptide in terms of scFv solubility and cell physiology.

“The pelB signal sequence resulted largely superior over the DsbA signal peptide in terms of scFv solubility and cell physiology [67].”

• cites [67] Kasli, 2019, Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: Selection of signal peptide and optimal growth conditions, AMB Express sourceSupported

Coexpression of an scFv with the periplasmic chaperones FkpA and Skp significantly improved the cytoplasmic solubility of the scFv and cell viability.

“In another study, the coexpression of an scFv with the periplasmic chaperones FkpA and Skp significantly improved the cytoplasmic solubility of the scFv and cell viability [168,172].”

• cites [168] Ow, 2010, Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production, Microb. Cell Fact. sourcePartly supported

  The Conclusions and Results and discussion report increased scFvD1.3 solubility and cell viability with Skp or FkpA co-expression. They describe periplasmic scFv expression, however, not improved cytoplasmic solubility. Page numbers were not provided in our copy. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [172] Wang, 2013, Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp, Front. Cell. Infect. Microbiol. sourcePartly supported

  The Results (page unavailable) report improved scFv solubility with Skp co-expression. The Introduction (page unavailable) mentions FkpA and increased cell viability only when describing earlier studies; this paper did not test FkpA co-expression or report improved viability, and its solubility result is not specifically localized to the cytoplasm. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Protein secretion can be effectively modulated at the transcriptional level by modifying the promoters in the expression vector.

“Protein secretion can be effectively modulated at the transcriptional level by modifying the promoters in the expression vector [145].”

• cites [145] Marschall, 2017, Tunable recombinant protein expression in E. coli: Promoter systems and genetic constraints, Appl. Microbiol. Biotechnol. sourcePartly supported

  The Introduction describes tuning recombinant protein expression in E. coli through promoter systems, and the prpBCDE operon section reports different expression levels with different promoter-based vectors. These passages concern protein expression, not secretion; the cited paper does not establish that modifying vector promoters effectively modulates secretion. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

The synergistic effect of DsbA/DsbC has been successfully assessed as an effective way to improve the soluble expression of Fabs and scFvs in the E. coli periplasmic space.

“The synergistic effect of DsbA/DsbC has been next successfully assessed as an effective way to improve the soluble expression of Fabs [56,88] and scFvs [170] in the E. coli periplasmic space.”

• cites [56] Ellis, 2017, Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab’ fragments, Biotechnol. Prog. sourceNot checked — no full text of the source
• cites [88] Rodriguez, 2017, Efficient antibody assembly in E. coli periplasm by disulfide bond. Folding factor Co-expression and culture optimization, Appl. Biochem. Biotechnol. sourceSupported
• cites [170] Sun, 2014, Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli, World J. Microbiol. Biotechnol. sourceNot checked — no full text of the source

A periplasmic recovery of 30 mg/L of functional Fab was reported using pelB and induction with 0.1 mM IPTG at 30 °C overnight when coexpressed with DsbA/C chaperones in shake flasks.

“A similar periplasmic recovery (30 mg/L) of functional Fab was reported using pelB and induction with 0.1 mM IPTG at 30 °C o.n. when coexpressed with the DsbA/C chaperones in shake flasks [88].”

• cites [88] Rodriguez, 2017, Efficient antibody assembly in E. coli periplasm by disulfide bond. Folding factor Co-expression and culture optimization, Appl. Biochem. Biotechnol. sourcePartly supported

  The Results section, “30 mg/L 3F3 Fab Yielded in Rich Culture Media” (page unavailable), reports 30 mg of purified Fab per liter at 30 °C. The Methods section, “3F3 Fab Expression” (page unavailable), describes pelB-based expression and overnight induction with 0.1 mM IPTG. However, the 30 mg/L condition used EnBase medium and co-expression with DsbA alone, not both DsbA and DsbC; our copy does not identify it as a shake-flask yield. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

The kappa light chain variable domains contain two conserved prolines in the cis conformation at positions L8 and L95, unlike the heavy-chain variable and lambda light chain variable antibody domains.

“Notably, the kappa light chain variable domains (Vκ) contain two conserved prolines in the cis conformation at positions L8 and L95 (Kabat numbering), unlike the heavy-chain variable (VH) and lambda light chain variable (Vλ) antibody domains [163].”

• cites [163] Johnson, 2001, Kabat database and its applications: Future directions, Nucleic Acids Res. sourceCould not verify

  The Introduction (page not provided) mentions Kabat numbering only for heavy-chain V-gene codons 1–94; the Results discuss nucleotide-sequence differences. Searches for cis prolines and for conserved prolines at L8 and L95 found no passage about those residues or a comparison of κ, λ, and heavy-chain domains. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Expression of an scFv antibody fragment using the novel vector pSAR-2 containing the pelB leader sequence and the rhamnose-inducible expression promoter obtained a yield of 1.2 g/L.

“Recently, the expression of an scFv antibody fragment has been used as a showcase for validating the efficacy of the novel vector pSAR-2 containing the pelB leader sequence and the rhamnose-inducible expression promoter, obtaining a yield of 1.2 g/L [71].”

• cites [71] Petrus, 2019, A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli, Appl. Microbiol. Biotechnol. sourceSupported

Rodriguez reported a significant improvement in functional Fab expression into the E. coli periplasm (30 mg/L) as a result of coexpression with wild type periplasmic DsbA/C.

“In this regard, Rodriguez and coworkers have reported a significant improvement in functional Fab expression into the E. coli periplasm (30 mg/L) as a result of its coexpression with the wild type periplasmic DsbA/C [88].”

• cites [88] Rodriguez, 2017, Efficient antibody assembly in E. coli periplasm by disulfide bond. Folding factor Co-expression and culture optimization, Appl. Biochem. Biotechnol. sourcePartly supported

  The Results section, “30 mg/L 3F3 Fab Yielded in Rich Culture Media” (page unavailable), reports 30 mg of purified Fab per liter. But Materials and Methods, “3F3 Fab Expression” (page unavailable), specifies that the optimized EnBase culture carried pBAD-DsbA, not a plasmid co-expressing both DsbA and DsbC. The claim gets the yield right but incorrectly attributes it to co-expression with both factors. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

The cytoplasmic variant of chaperone FkpA (cyt-FkpA) was successfully used for improving the periplasmic expression of a Fab fragment.

“In another report, the cytoplasmic variant of chaperone FkpA (cyt-FkpA) was sucessfully used for improving the periplasmic expression of a Fab fragment [169].”

• cites [169] Levy, 2013, Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm, J. Immunol. Methods sourceSupported

A recovery of 10 mg/mL of highly immunoreactive Fabs was obtained using pelB, the BL21 strain and the SB medium in combination with a harmonizing DNA approach.

“In another study, a recovery of 10 mg/mL of highly immunoreactive Fabs was obtained by using pelB, the BL21 strain and the SB medium in combination with a harmonizing DNA approach [60].”

• cites [60] Kulmala, 2017, Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli, Sci. Rep. sourcePartly supported

  The Methods (Up-scaled protein production) describe harmonized Fab0 produced in BL21 using SB medium, and the Results (Characteristics of the parent genes and the genetic variants) identify a PelB signal sequence. But the Results (Up-scaled protein production) report 11.0 and 9.5 mg/L of functional Fab—not 10 mg/mL. The claimed concentration is 1,000 times higher than the approximately 10 mg/L reported. (LLM, conf=0.91)

  Experimental — accuracy not yet measured

Combinatorial screening of different signal peptides in a titratable system that tunes protein production rates was a valuable and effective approach to enhance scFv recombinant production yields in the periplasm of E. coli.

“Karyolaimos and coworkers [68] have successfully demonstrated that a combinatorial screening of different signal peptides in a titratable system that tunes protein production rates was a valuable and effective approach to enhance scFv recombinant production yields in the periplasm of E. coli.”

• cites [68] Karyolaimos, 2019, Enhancing recombinant protein yields in the E. coli periplasm by combining signal. Peptide and production rate screening, Front. Microbiol. sourceSupported

Correct folding of scFv and Fab fragments is highly dependent on the activity of PPIases.

“In particular, the correct folding of scFv and Fab fragments has been found to be highly dependent on the activity of PPIases [162].”

• cites [162] Feige, 2010, How antibodies fold, Trends Biochem. Sci. sourcePartly supported

  The section ‘From the folding of antibody domains to complete IgG molecules’ (page unavailable) says that proline isomerization is rate-limiting for CH1 folding and relates this step to Fab folding. The concluding remarks also say that peptidyl-prolyl isomerization controls antibody folding. Neither passage establishes that correct folding of both scFv and Fab fragments is highly dependent on PPIase enzyme activity; a requirement for isomerization is not itself evidence of dependence on those enzymes. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The slow isomerization of the peptide bond preceding Pro-L95 is important because it must be in the cis conformation for formation of the native VH/VL interface.

“Pioneering studies reported by Plückthun and coworkers [164,165] on the aggregation properties of scFv fragments, demonstrated that the slow isomerization of the peptide bond preceding Pro-L95 is important because it must be in the cis conformation for the formation of the native VH/VL interface.”

• cites [164] Ramm, 1999, Removal of the conserved disulfide bridges from the scFv fragment of an antibody: Effects on folding kinetics and aggregation, J. Mol. Biol. sourceNot checked — no full text of the source
• cites [165] Jager, 1997, The rate-limiting steps for the folding of an antibody scFv fragment, FEBS Lett. sourceSupported

The highest periplasmic production yield of an scFv was 0.2 g/L of culture, achieved using the OmpA signal peptide, LB medium and induction with 100 μM rhamnose at 30 °C in a shake flask.

“The highest periplasmic production yield of an scFv was 0.2 g/L of culture and was achieved using the OmpA signal peptide, LB medium and induction with 100 μM rhamnose at 30 °C in a shake flask [68].”

• cites [68] Karyolaimos, 2019, Enhancing recombinant protein yields in the E. coli periplasm by combining signal. Peptide and production rate screening, Front. Microbiol. sourceSupported

The three main antibody surrogates, single-domain antibodies, scFv and Fabs, are continuously developed and reformatted into bispecific/multi-specific molecules or cytotoxic/radioactive drug carriers for clinical applications.

“Thanks to their improved pharmacokinetics and their structural and functional flexibility, the three main antibody surrogates, single-domain antibodies (dAbs), scFv and Fabs, are continuously developed and reformatted into bispecific/multi-specific molecules or cytotoxic/radioactive drug carriers to achieve a desired biological efficacy and for multiple clinical applications [1].”

• cites [1] Bates, A., and Power, A.C. (2019). David vs. goliath: The structure, function, and clinical prospects of antibody fragments. Antibodies, 8. sourcePartly supported

  The Introduction (section 1) identifies Fab, scFv and single-domain antibodies as modular fragments that can be linked into bispecific or multispecific molecules. The Oncology section (4.1.6) reports toxin and radioisotope conjugates with clinical applications. But the citing sentence’s blanket claim of improved pharmacokinetics is not supported: the Introduction says these fragments’ smaller size also causes faster renal excretion, potentially requiring higher or more frequent doses. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Using the pSAR2 plasmid with a rhamnose promoter, the scFv PGT135 antibody fragment was successfully produced in the periplasmic space, with amounts up to about 1.2 g/L of biologically active scFv PGT135 recovered.

“In particular, by using the pSAR2 plasmid with a rhamnose promoter, the scFv PGT135 antibody fragment was successfully produced in the periplasmic space. Amounts up to about 1.2 g/L of the biologically active scFv PGT135 were recovered [71].”

• cites [71] Petrus, 2019, A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli, Appl. Microbiol. Biotechnol. sourcePartly supported

  The Results and Discussion report that the rhamnose-inducible pSAR-2 vector produced PGT135 scFv directed to the periplasm, and that the purified fragment was biologically active. But the approximately 1.2 g/L figure (1182 ± 2 mg/L) is the amount measured in total cells in shaker flasks, not the amount of active antibody recovered: about 0.5 g/L was soluble, and the Discussion reports about 58 mg/L purified. (LLM, conf=0.93)

  Experimental — accuracy not yet measured

The Ecobody technology enables efficient and cost-effective production of functional proteins including monoclonal antibodies and related fragments.

“The well-known and innovative Ecobody technology [179] enables an efficient and cost-effective production of functional proteins including monoclonal antibodies and related fragments [76,180,181,182].”

• cites [179] Nagai, 2017, Ecobody technology: Rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation, Sci. Rep. sourceSupported
• cites [76] Ojima-Kato, T., Morishita, S., Uchida, Y., Nagai, S., Kojima, T., and Nakano, H. (2018). Rapid generation of monoclonal antibodies from single B cells by ecobody technology. Antibodies, 7. sourceSupported
• cites [180] Jiang, 2006, A novel strategy for generation of monoclonal antibodies from single B cells using rt-PCR technique and in vitro expression, Biotechnol. Prog. sourceNot checked — no full text of the source
• cites [181] Sabrina, 2010, In vitro generation of anti-hepatitis B monoclonal antibodies from a single plasma cell using single-cell RT-PCR and cell-free protein synthesis, J. Biosci. Bioeng. sourceNot checked — no full text of the source
• cites [182] Hashimura, 2015, In vitro generation of rabbit anti-Listeria monocytogenes monoclonal antibody using single cell based RT-PCR linked cell-free expression systems, J. Immunol. Methods sourceNot checked — no full text of the source

There is no universal E. coli-based methodology for efficient production of antibody fragments.

“it is evident that there is not a universal E. coli-based methodology for their efficient production”

Contributions to adapting E. coli machinery to antibody fragment production have come from structural biology and bioinformatics in addition to advanced molecular genetics, basic biology and chemical biology.

“a contribution has been also provided by structural biology and bioinformatics in addition to advanced molecular genetics, basic biology and chemical biology.”

In the presence of short linkers, one antibody fragment's VL or VH domain interacts with another molecule's complementary domain through domain swapping, generating dimers or higher-order oligomers.

“In the presence of short linkers, one antibody fragment’s VL or VH domain interacts with another molecule’s complimentary domain through a mechanism known as “domain swapping” generating dimers or higher-order oligomers [21].”

• cites [21] Worn, 2001, Stability engineering of antibody single-chain Fv fragments, J. Mol. Biol. sourceNot checked — no full text of the source

The absence of the Fc domain and the small size of antibody fragments results in a shorter half-life compared to full-length antibodies.

“On the other hand, the absence of the Fc domain and the small size results in a shorter half-life compared to full-length antibodies [2].”

• cites [2] Kholodenko, 2017, Isolation of induced pluripotent cells from stromal liver cells of patients with alcoholic cirrhosis, Bull. Exp. Biol. Med. sourceNot checked — no full text of the source

Practical use of scFv fragments is limited by aggregation propensity and consequent low homogeneity due to dynamic structural features (open and closed state) depending on interchain VH-VL interactions.

“Despite the relatively simple structure of scFv fragments, their practical use is limited by the aggregation propensity and consequent low homogeneity. This is due to their dynamic structural features (open and closed state) depending on interchain VH-VL interactions.”

Replacement of residues vH44 and vL100 leads to one of the most favorable disulfide configurations at the VH-VL interface.

“It has been found that the replacement of residues vH44 and vL100 leads to one of the most favorable disulfide configurations [33].”

• cites [33] Weatherill, 2012, Towards a universal disulphide stabilised single chain Fv format: Importance of interchain disulphide bond location and vL-vH orientation, Protein Eng. Des. Sel. sourceNot checked — no full text of the source

The E. coli periplasm provides natural oxidizing conditions for disulfide bond formation and isomerization due to enzymes and specific chaperones and foldases.

“The E. coli periplasm provides the natural oxidizing conditions for disulfide bond formation and isomerization due to the presence of enzymes and specific chaperones and foldases that facilitate the production of soluble and active proteins containing disulfide bonds [130].”

• cites [130] Choi, 2004, Secretory and extracellular production of recombinant proteins using Escherichia coli, Appl. Microbiol. Biotechnol. sourceNot checked — no full text of the source

Use of the SHuffle strain in combination with different chaperones or fusion tags and with Enpresso B medium paves the way to large-scale production of antibody fragments through cytoplasmic expression on a lab scale.

“Its use in combination with different chaperones or fusion tags and with the Enpresso B medium, therefore, paves the way to the large-scale production of antibody fragments through cytoplasmic expression also on a lab scale [128].”

• cites [128] Ren, 2016, Use of the SHuffle strains in production of proteins, Curr. Protoc. Protein Sci. sourceNot checked — no full text of the source

Use of redox-altered mutants of strains such as SHuffle and Origami restores an oxidizing environment in bacterial cytoplasm, thereby significantly facilitating production of soluble cytoplasmic recombinant proteins containing disulfide bonds.

“The use of redox-altered mutants of strains such as SHuffle and Origami and others restores an oxidizing environment in the bacterial cytoplasm, thereby their use significantly facilitates the production of soluble cytoplasmic recombinant proteins containing disulfide bonds.”

Protein expression using pBAD and pRha vectors is more tightly controlled compared to other expression systems such as pET vectors.

“Protein expression using pBAD and pRha vectors is more tightly controlled compared to other expression systems such as pET vectors.”

A Fab fragment bearing a leucine zipper pair at the C-termini of both heavy and light chains, named Zipbody, enhances chain pairing in the active form in E. coli cytoplasm expression and in cell-free protein synthesis systems.

“They also described an ad hoc engineered Fab fragment bearing a leucine zipper (LZ) pair at the C-termini of both the heavy and light chains, a construct named Zipbody. These extensions would enhance the chain pairing in the active form in both the E. coli cytoplasm expression and in the cell-free protein synthesis systems, a technology named Ecobody [124,125,126].”

• cites [124] Nagai, 2017, N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae, J. Biosci. Bioeng. sourceNot checked — no full text of the source
• cites [125] Ritthisan, 2018, SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm, J. Biosci. Bioeng. sourceNot checked — no full text of the source
• cites [126] Fukui, 2016, ‘Zipbody’ leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems, Protein Eng. Des. Sel. sourceNot checked — no full text of the source

Production of soluble cyclic scFv (2.8 mg/L) in bacterial cytoplasm was optimized by combination of chaperone coexpression and SHuffle strain.

“Accordingly, they optimized the production of soluble cyclic scFv (2.8 mg/L) in bacterial cytoplasm by a combination of chaperone coexpression and SHuffle strain [36].”

• cites [36] Liu, C., Kobashigawa, Y., Yamauchi, S., Fukuda, N., Sato, T., Masuda, T., Ohtsuki, S., and Morioka, H. (2020). Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression. J. Biochem. sourceNot checked — no full text of the source

As a consequence of more stringent regulation of target gene expression, attainable yields using pBAD and pRha are relatively lower compared to those reached using pET systems.

“As a consequence of more stringent regulation of target gene expression, the attainable yields are relatively lower compared to those reached using pET systems.”

The SKIK sequence (serine-lysine-isoleucine-lysine) improves the cytoplasmic expression of Fab fragments in the E. coli SHuffle T7 Express strain using LB medium at 16 °C for 24 h.

“Recently, the research group of Nakano has reported the use of a small tag, referred as the SKIK sequence (serine-lysine-isoleucine-lysine), that is able to improve the cytoplasmic expression of Fab fragments in the E. coli SHuffle T7 Express strain using the LB medium at 16 °C for 24 h [124,125].”

• cites [124] Nagai, 2017, N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae, J. Biosci. Bioeng. sourceNot checked — no full text of the source
• cites [125] Ritthisan, 2018, SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm, J. Biosci. Bioeng. sourceNot checked — no full text of the source

The anti-HER2 scFv yield with CyDisCo was 70.5% higher than that obtained in SHuffle using LB medium (147 mg/L).

“and 70.5% higher than that obtained in SHuffle using the LB medium (147 mg/L) [109].”

• cites [109] Ahmadzadeh, 2020, Anti-HER2 scFv expression in Escherichia coli SHuffle((R))T7 express cells: Effects on solubility and biological activity, Mol. Biotechnol. sourceNot checked — no full text of the source

The SHuffle strain has enormous potential for production of soluble fragments of active antibodies in the E. coli cytoplasm, especially scFv.

“Collectively, these results highlight the enormous potential of the SHuffle strain for the production of soluble fragments of active antibodies in the E. coli cytoplasm, especially scFv.”

Liu and coworkers reported cytoplasmic expression of a soluble and active scFv with yield up to 12.8 mg/L by mimicking oxidizing environment with SHuffle strain and coexpression of chaperones GroEL, GroES, DnaK, DnaJ, GrpE and trigger factor.

“Recently, Liu and coworkers [102] have reported the cytoplasmic expression of a soluble and active scFv with a yield up to 12.8 mg/L. They have strategically mimicked the oxidizing environment by a combination of the SHuffle strain and by the coexpression of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE and trigger factor (TF) [102].”

• cites [102] Liu, 2019, Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization, J. Biochem. sourceNot checked — no full text of the source

Sun reported a yield of 33 mg/L of a soluble and active scFv in shaking-flask cultures using BL21 (DE3) cells, 2xYT media and induction with 0.2 mM IPTG for 4 h at 30 °C.

“Sun and coworkers reported a yield of 33 mg/L of a soluble and active scFv in shaking-flask cultures by using BL21 (DE3) cells, 2xYT media and following induction with 0.2 mM IPTG for 4 h at 30 °C [170].”

• cites [170] Sun, 2014, Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli, World J. Microbiol. Biotechnol. sourceNot checked — no full text of the source

Fab fragments bearing the OmpA signal peptide were efficiently produced as soluble periplasmic products by coexpression with DnaK/DnaJ/GrpE in shake flasks using LB medium and inducing with 0.1 mM IPTG at 25 °C for 8 h.

“Additionally, Fab fragments bearing the OmpA signal peptide were efficiently produced as soluble periplasmic products by coexpression with DnaK/DnaJ/GrpE in shake flasks using the LB medium and inducing with 0.1 mM IPTG at 25 °C for 8 h [99].”

• cites [99] Dariushnejad, 2019, Effect of DnaK/DnaJ/GrpE and DsbC Chaperons on Periplasmic Expression of Fab Antibody by E. coli SEC Pathway, Int. J. Pept. Res. Ther. sourceNot checked — no full text of the source

The secretion efficiency of the STII signal peptide could be further improved by coexpression of TolC, the major efflux pump in gram-negative bacteria.

“Of interest, they found that the secretion efficiency of the STII signal peptide could be further improved by the coexpression of TolC, the major efflux pump in gram-negative bacteria.”

Ellis developed a novel approach based on coexpression of Fab fragments with DsbC in engineered E. coli Tsp spr strains, achieving a yield of periplasmic Fab over 2.4 g/L in fermentation scale after 40 h post-induction.

“Similarly, Ellis and coworkers developed a novel approach based on the coexpression of Fab fragments with DsbC in ad hoc engineered E. coli strain (Tsp spr strains) achieving a very high yield of periplasmic Fab approaching over 2.4 g/L in fermentation scale after 40 h post-induction [56].”

• cites [56] Ellis, 2017, Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab’ fragments, Biotechnol. Prog. sourceNot checked — no full text of the source

Supplementing a nitrogen source at low temperature is critical for Fab productivity in E. coli fermentations.

“These results indicate that supplementing a nitrogen source at low temperature is critical for Fab productivity in E. coli fermentations [90].”

• cites [90] Kim, 2018, Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source, Enzyme Microb. Technol. sourceNot checked — no full text of the source

Use of the heat-stable enterotoxin II signal peptide led to an expression yield of 332 mg/L of soluble Fab.

“In another study, the use of the heat-stable enterotoxin II (STII) signal peptide led to an expression yield of 332 mg/L of soluble Fab.”

The pACYC-Duet-Dsb coexpression vector might be a useful tool for the production of soluble and functional scFv antibody fragments.

“These results suggested that the pACYC-Duet-Dsb coexpression vector might be a useful tool for the production of soluble and functional scFv antibody fragments.”

Overexpression of TolC dramatically enhanced Fab recovery from the extracellular medium.

“Therefore, the overexpression of TolC dramatically enhanced the Fab recovery from the extracellular medium.”

A yield of 25 mg/L of a periplasmic Fab fragment in shake flasks was obtained using the rhamnose-inducible promoter and the mal and pelB secretion sequences for the heavy and light chains, respectively.

“Kumar and coworkers have obtained a yield of 25 mg/L of a periplasmic Fab fragment in shake flasks by using the rhamnose-inducible promoter (rhaBAD) and the mal and pelB secretion sequences for the heavy and light chains, respectively.”

The RiboTite gene expression control is based on dual transcriptional-translational gene expression control where a dual Lac-operator/repressor promoter and an orthogonal riboswitch modulate both T7 RNAP and the gene of interest.

“It is based on the dual transcriptional–translational gene expression control where a dual Lac-operator/repressor promoter and an orthogonal riboswitch modulate both T7 RNAP and the GOI.”

Coexpression of the pKJE7 plasmid containing GrpE/DnaK/DnaJ had the highest outcome, up to 50%, on solubility compared to other chaperone combinations.

“Importantly, the coexpression of the pKJE7 plasmid containing GrpE/DnaK/DnaJ had the highest outcome (up to 50%) on solubility compared to other chaperone combinations.”

Use of an optimal nucleotide triplet coding for leucine at position 5 of the pelB sequence of the light chain resulted in a reduction of the expression level.

“The use of an optimal nucleotide triplet coding for leucine in position 5 of the pelB sequence of the light chain resulted in a reduction of the expression level.”

Use of the alkaline phosphatase promoter in combination with the heat-stable enterotoxin II signal peptide is a promising strategy for extracellular production of Fab fragments, reaching up to 10 mg/L.

“They have demonstrated that the use of the alkaline phosphatase promoter (phoA) in combination with the heat-stable enterotoxin II (STII) signal peptide (phoA-STII system) is a promising strategy for the extracellular production of Fab fragments, reaching up to 10 mg/L [160].”

• cites [160] Luo, 2019, A general platform for efficient extracellular expression and purification of Fab from Escherichia coli, Appl. Microbiol. Biotechnol. sourceNot checked — no full text of the source

Coexpression of the periplasmic chaperone DsbC significantly increased Fab antibody fragment expression in the bacterial cytoplasm using the Origami (DE3) strain.

“As already mentioned above in the cytoplasmic expression paragraph, it has been reported that the coexpression of the periplasmic chaperone DsbC significantly increased the Fab antibody fragment expression in the bacterial cytoplasm using the Origami (DE3) strain [113].”

• cites [113] Levy, 2001, Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones, Protein Expr. Purif. sourceNot checked — no full text of the source

When the same Fab was coexpressed with DsbC in fermentation scale using an engineered E. coli strain deficient in Tsp and SRP, an optimal recovery of 2.4 g/L was reached.

“When the same Fab was coexpressed with DsbC in fermentation scale using the ad hoc genetically modified E. coli strain deficient in the Tail specific protease (Tsp) and SRP, an optimal recovery of 2.4 g/L was reached [56].”

• cites [56] Ellis, 2017, Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab’ fragments, Biotechnol. Prog. sourceNot checked — no full text of the source

The cyt-FkpA strategy improved the levels of soluble periplasmic Fab from 0.4-2.5 mg/L to 3.5-14.2 mg/L.

“The strategy improved the levels of soluble periplasmic Fab from 0.4–2.5 mg/L to 3.5–14.2 mg/L.”

A functional recombinant scFv was efficiently recovered (2.86 mg/L) from the extracellular medium by adding 0.25% Triton X-100.

“In this way, a functional recombinant scFv has been efficiently recovered (2.86 mg/L) from the extracellular medium by adding 0.25% Triton X-100 [159].”

• cites [159] Na, 2019, Extracellular production of functional single-chain variable fragment against aflatoxin B1 using Escherichia coli, Lett. Appl. Microbiol. sourceNot checked — no full text of the source

The alkaline phosphatase promoter and the heat-stable enterotoxin II leader sequence have been described as a positive combination to facilitate E. coli extracellular production of Fab fragments.

“Of note, the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence have been also described as a positive combination to facilitate the E. coli extracellular production of Fab fragments [160].”

• cites [160] Luo, 2019, A general platform for efficient extracellular expression and purification of Fab from Escherichia coli, Appl. Microbiol. Biotechnol. sourceNot checked — no full text of the source

Coexpression of DnaK/DnaJ/GrpE results in a 2.5-fold increase in the periplasmic expression level of an anti-TNF-α Fab in shake flasks using LB medium and BL21(DE3) strains.

“For instance, Dariushnejad and coworkers have reported that the coexpression of DnaK/DnaJ/GrpE (DnaKJE) results in a 2.5-fold increase in the periplasmic expression level of an anti-TNF-α Fab in shake flasks, using LB medium and BL21(DE3) strains [99,100].”

• cites [99] Dariushnejad, 2019, Effect of DnaK/DnaJ/GrpE and DsbC Chaperons on Periplasmic Expression of Fab Antibody by E. coli SEC Pathway, Int. J. Pept. Res. Ther. sourceNot checked — no full text of the source
• cites [100] Farajnia, 2020, Effect of molecular chaperone on the soluble expression of recombinant fab fragment in E. coli, Int. J. Pept. Res. Ther. sourceNot checked — no full text of the source

Optimization of extracellular secretion is becoming another valid option to produce active folded recombinant antibody fragments in E. coli.

“However, the optimization of extracellular secretion is becoming another valid option to produce active folded recombinant antibody fragments in E. coli.”

The choice of signal peptide has a strong impact on recombinant protein production rate and yield in the periplasm.

“The choice of the signal peptide has a strong impact on recombinant protein production rate and yield in the periplasm.”

No relevant improvements were detected using the Shuffle strain in the DnaKJE coexpression study.

“No relevant improvements were detected using the Shuffle strain.”

The ESETEC Wacker secretion technology relies on the use of a K12 modified strain that enables yields up to 4.0 g/L for Fab fragments and 3.5 g/L for scFvs into the fermentation broth.

“It relies on the use of the K12 modified strain that enables yields up to 4.0 g/L for Fab fragments and 3.5 g/L for scFvs into the fermentation broth (https://www.wacker.com/cms/en-us/products/brands/esetec/esetec.html).”

The Xpress CF technology has been used to produce Sutro's clinical ADC products STRO-001 and STRO-002, in which a cytotoxin is conjugated to an antibody containing non-natural amino acids.

“For example, the technology has been used to produce the Sutro’s clinical ADC products STRO-001 and STRO-002, in which a cytotoxin is conjugated to an antibody containing non-natural amino acids (STR001—Clinical Trial: NCT03424603. Available online: https://clinicaltrials.gov/ct2/show/NCT03424603).”

A trial and error optimization process is necessary for determining successful experimental settings and achieving scalability of antibody fragment expression processes at an industrial level.

“therefore, a trial and error optimization process is necessary for the determination of the successful experimental settings and to achieve scalability of antibody fragment expression processes at an industrial level.”

The Ecobody technology has been successfully employed to produce two Fab fragments needed to set up a rapid ELISA assay for detection of swine influenza virus.

“Recently, the Ecobody technology has been successfully employed to produce two Fab fragments needed to set up a rapid ELISA assay for the detection of swine influenza virus [183].”

• cites [183] Chertchinnapa, 2020, Development of a dual monoclonal antibody sandwich enzyme-linked immunosorbent assay for the detection of swine influenza virus using rabbit monoclonal antibody by Ecobody technology, J. Biosci. Bioeng. sourceNot checked — no full text of the source

Antibody fragments such as Fabs and scFvs can be conveniently prepared in E. coli backgrounds.

“antibody fragments like Fabs, scFvs and other similar surrogates that lack the glycosylation, can be conveniently prepared in E. coli backgrounds.”

• AssumptionRests on an assumption: Antibody fragments such as Fabs and scFvs lack glycosylation.