Effects of variable domain orientation on anti‐HER2 single‐chain variable fragment antibody expressed in the Escherichia coli cytoplasm

Abstract

Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

Ribosome display ofN-linked glycoproteins in cell-free extracts

“…3c ). Nearly identical results were obtained when CFGpS reactions were primed with plasmid DNA encoding a different acceptor POI, namely an scFv antibody specific for human epidermal growth factor receptor 2 (scFv-HER2) that was modified at its C-terminus with a DQNAT glycosylation tag 14,43 ( Fig. 3b and c ).…”

Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts

“…Importantly, the mRNA encoding SecM17-stalled Im7 N58 was stably attached to ribosomes, as evidenced by the prominent amplicon generated via RT-PCR of ribosome-associated RNA (Figure 3c). Nearly identical results were obtained when CFGpS reactions were primed with plasmid DNA encoding a different POI, namely, an scFv antibody specific for human epidermal growth factor receptor 2 (scFv-HER2) that was modified at its C-terminus with a DQNAT glycosylation tag 6,24 (Figure 3b,c). Using immobilized HER2 as the antigen, we selected glycoRNC complexes displaying glycosylated scFv-HER2 DQNAT −SecM17 and observed that the encoding mRNA remained associated with these functionally selected glycoRNCs (Figure 3d).…”

Expression, purification and characterisation of a human anti-CDK4 single-chain variable fragment antibody

“…Expression of soluble antibody proteins is the first required step for determining antibody protein quality and functional activity. In fact, a large number of studies have been focused on improving expression of soluble and stable scFv antibodies using various bacterial and yeast expression systems [ 20 , 39 – 41 ]. Among existing expression systems, those based on E. coli have become popular for use in scFvs preparation, although use of an E. coli -based system to generate the two intrachain disulfide bonds necessary for antibody folding and antigen-binding activity can be challenging [ 20 ].…”

“…In fact, a large number of studies have been focused on improving expression of soluble and stable scFv antibodies using various bacterial and yeast expression systems [ 20 , 39 – 41 ]. Among existing expression systems, those based on E. coli have become popular for use in scFvs preparation, although use of an E. coli -based system to generate the two intrachain disulfide bonds necessary for antibody folding and antigen-binding activity can be challenging [ 20 ]. To address this problem, researchers have designed various strategies to produce functional scFvs by promoting their accumulation within the periplasm, where the oxidising environment supports disulfide bond formation and corrects scFv folding [ 20 , 37 , 42 – 45 ].…”

“…Among existing expression systems, those based on E. coli have become popular for use in scFvs preparation, although use of an E. coli -based system to generate the two intrachain disulfide bonds necessary for antibody folding and antigen-binding activity can be challenging [ 20 ]. To address this problem, researchers have designed various strategies to produce functional scFvs by promoting their accumulation within the periplasm, where the oxidising environment supports disulfide bond formation and corrects scFv folding [ 20 , 37 , 42 – 45 ]. In this work, AK2 was expressed in soluble form in E. coli HB2151 cells after it was isolated from the scFv phage library.…”