Gąciarz A, Ruddock LW (2017) Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS ONE 12(12): e0189964. https://doi.org/10.1371/journal.pone.0189964
Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv
|Author:||Gąciarz, Anna1; Ruddock, Lloyd W.1|
1Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
|Online Access:||PDF Full Text (PDF, 2.1 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe201902205698
Public Library of Science,
|Publish Date:|| 2019-02-20
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.
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
1182 Biochemistry, cell and molecular biology
This work was supported by Academy of Finland (http://www.aka.fi/en; LWR), Sigrid Juselius Foundation (http://sigridjuselius.fi/en/main-page/; LWR), Biocenter Oulu (http://www.oulu.fi/biocenter/; LWR), Magnus Ehrnrooth Foundation (https://www.magnusehrnroothinsaatio.fi/en/; AG) and University of Oulu Scholarship Foundation (http://www.oulu.fi/university/node/38384; AG).
© 2017 Gąciarz, Ruddock. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.