Heterologous production and structural studies for disulfide rich proteins

Escherichia coli (E. coli) has been considered as one of the best expression hosts for the production of eukaryotic recombinant proteins. However, the production of disulfide-containing proteins in prokaryotic hosts like E. coli has always been challenging. Many strategies including different strains, chaperone co-expression and low temperature induction, has been used to overcome the issue of insoluble protein production. The development of CyDisCo™ (cytoplasmic disulfide bond formation in E. coli), facilitated and enhanced the production of disulfide rich eukaryotic proteins expressed in the cytoplasm of E. coli. CyDisCo™ expression technology uses co- or pre-expression of a sulfhydryl oxidase for de novo disulfide bond formation and a protein disulfide isomerase for the catalysis of disulfide bond isomerization.
In this study, we attempted to test the limits of E. coli using CyDisCo™ in the manufacturing of basement membrane (BM) proteins, including perlecan, nidogen-2 and fibulin-2. Other disulfide-containing proteins like growth factor PDGF-BB and enzymes such as CalB and MTP were also studied. The ultimate objective of the study was to understand the biological significance of these proteins by solving their structures at atomic level using x-ray crystallography.
Basement membrane proteins have been intensively studied at a cellular level. Unfortunately, limited structural information of BM proteins at the atomic level is available. One of the reasons for this is issues related to protein production due to them being disulfide rich. Protein fragments of BM proteins were constructed, and expression tests were carried out in an E. coli host using CyDisCo™. The successful production of soluble BM protein fragments covered approximately 84 %, 32 % and 61 % for perlecan, nidogen-2 and fibulin-2, respectively (having 83 %, 40 % and 79 % of the total disulfide bonds). In the study, biochemical, biophysical, and structural characterization was carried out for the purified protein fragments. Among the results obtained, the soluble production of mouse perlecan region 3 residues G503-T1672, ~127 kDa in size having 44 disulfide bonds, was an exceptional and novel achievement of E. coli using CyDisCo™. Furthermore, the first crystal structures of a ~40 kDa protein fragment from perlecan region 3 and a protein fragment from a fibulin-2 forming homodimer, ~26 kDa in size having 17 disulfide bonds, were also solved during the study.
Additionally, the mature form of other eukaryotic disulfide-containing proteins including MTP, CalB and PDGF-BB were successfully produced in E. coli. This work included structure-function studies of the enzymes.