Abstract
CyDisCo is a system facilitating disulfide bond formation in recombinant proteins in the cytoplasm of Escherichia coli. Previously we screened for soluble expression of single chain antibody fragments (scFv) in the cytoplasm of E. coli in the presence and absence of CyDisCo, with >90% being solubly expressed. Two scFv, those derived from natalizumab and trastuzumab, were solubly produced in high amounts even in the absence of folding catalysts i.e. disulfide bond formation is not critical for their folding.Here we investigate the contribution of the framework and the complementarity determining regions (CDRs) of scFv to the disulfide-independence of folding. We swapped CDRs between four scFv that have different properties, including two scFv that can efficiently fold independently from disulfide bonds and two more disulfide-dependent scFv. To confirm disulfide-independence we generated cysteine to alanine mutants of the disulfide-independent scFv. All of the scFv were tested for soluble expression in the cytoplasm of E. coli in the presence and absence of the oxidative folding catalysts Erv1p and PDI.Eight of the hybrid scFv were solubly produced in the presence of CyDisCo, while seven were solubly produced in the absence of CyDisCo, though the yields were often much lower when CyDisCo was absent. Soluble expression was also observed for scFv natalizumab and trastuzumab containing no cysteines. We compared yields, thermal stability and secondary structure of solubly produced scFv and undertook binding studies by western blotting, dot blotting or surface plasmon resonance of those produced in good yields. Our results indicate that both the CDRs and the framework contribute to the disulfide-dependence of soluble production of scFv, with the CDRs having the largest effect. In addition, there was no correlation between thermal stability and disulfide-dependence of folding and only a weak correlation between the yield of protein and the thermal stability of the protein.
Highlights
Antibodies are tetrameric proteins consisting of two heavy and two light chains that are held together by inter-chain disulfide bonds
To examine the contribution of the framework and the complementarity determining regions (CDRs) to disulfide independent folding we chose four scFv from our previous study all of which could be assayed for binding activity
Trastuzumab and natalizumab were chosen as the only examples we have found experimentally to date to show efficient disulfide-independent folding in the cytoplasm of E. coli in deep well plates
Summary
Expression vectors (Table 1) were made by standard molecular biology techniques. The scFv expression vector used was based on pET23 in which the T7 promoter was replaced with tac promoter, as described previously [27]. All vectors encoded VL and VH in a VL-(Gly4Ser)3VH-GSH6 format. The trastuzumab, natalizumab, IgA1 and Maa scFv were constructed and described previously (24). ScFv with swapped CDRs along with trastuzumab and natalizumab scFv with all cysteines mutated to alanine were synthetized by GenScript and cloned Nde I / BamH I into an expression vector in frame with a C-terminal GSH6-tag. For scFv with swapped CDRs the framework residue 71 of both the VH and VL domains of the hybrid were matched with the corresponding residue of the CDR donor as these framework residues have been implicated in preserving the structure and function of the CDRs [24, 30, 31]
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