Resolving the challenge of insoluble production of mature human growth differentiation factor 9 protein (GDF9) in E. coli using bicistronic expression with thioredoxin

Abstract

Growth differentiation factor 9 (GDF9) is an oocyte-derived protein with fundamental functions in folliculogenesis. While the crucial contributions of GDF9 in follicular survival have been revealed, crystallographic studies of GDF9 structure have not yet been carried out, essentially due to the insoluble expression of GDF9 in E. coli and lack of appropriate source for structural studies. Therefore, in this study, we investigated the impact of different expression rate of bacterial thioredoxin (TrxA) using bicistronic expression constructs to induce the soluble expression of mature human GDF9 (hGDF9) driven by T7 promoter in E. coli. Our findings revealed that in BL21(DE3), the high rate of TrxA co-expression at 30 °C was sufficiently potent for the soluble expression of hGDF9 and reduction of inclusion body formation by 4 fold. We also successfully confirmed the bioactivity of the purified soluble hGDF9 protein by evaluation of follicle-stimulating hormone receptor gene expression in bovine cumulus cells derived from small follicles. This study is the first to present an effective approach for expression of bioactive form of hGDF9 using TrxA co-expression in E. coli, which may unravel the current issues regarding structural analysis of hGDF9 protein and consequently provide a better insight into hGDF9 functions and interactions.

Keywords: Bicistronic gene expression system; Bovine cumulus cells; Follicle-stimulating hormone receptor; Human growth differentiation factor 9; Inclusion body; Thioredoxin co-expression.