Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization

Abstract

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

“…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]. Through a systematic screening of chaperones, they have identified the GroEL-GroES system as the best performing for the preparation of scFv fragments cloned in pET28 and bearing an N-terminus hexahistidine tag.…”

“…Stress minimization results in the increased viability of cells and process robustness [70,94]. In the so-called Design of Experiments (DoE) setting [95] the optimization of the expression of recombinant Fab and scFv fragments in stress minimization conditions has been successfully achieved through the selection of media [70,88,96,97], screening of signal peptide sequences [67,68,75,98] and optimization of co-expressing chaperone proteins [56,88,[99][100][101][102]. 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].…”

“…Some comparative studies have demonstrated that the levels of functional Fabs and scFvs have been largely improved in the presence of these cytoplasmic chaperones [113,114]. 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].…”

Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

“…Redox engineered E. coli SHuffle cells have been previously demonstrated to be an attractive platform for the expression of various antibody formats, such as full-length IgG (Reddy et al 2018 ; Robinson et al 2015 ), Fab’ fragments (Abe et al 2014 ; Mori et al 2018 ; Yusakul et al 2018 ), scFv chains (Ahmadzadeh et al 2020 ; Liu et al 2019 ; Vermeulen et al 2018 ) and VHH domains (Eliseev et al 2018 ; Ta et al 2015 ; Zarschler et al 2013 ). Although SHuffle cells lack the eukaryotic glycosylation machinery, by engineering mutations in the Fc region of antibodies, the requirement for glycosylation for efficient binding of the IgG to its cognate receptor was bypassed (Robinson et al 2015 ).…”

Anti-CXCR4 Single-Chain Variable Fragment Antibodies Have Anti-Tumor Activity

“…Lack of Fc can decrease its efficacy for cancer therapy, but scFv can be modified in different ways to increase its efficacy for therapeutic use as described below. scFv antibody comprises a variable region of heavy chain and a variable region of light chain, and the two chains are connected by a short peptide linker (25). It can act as an antagonist to block tumor growth signaling, induce tumor cell apoptosis and suppress tumor growth (22).…”