Integrated in silico structural design & upstream expression optimization of an A7G mutant rh-G-CSF.

Abstract

The study focused on the rational design of recombinant human G-CSF (rhG-CSF) by structure-based single-site modification and process optimization. The research was conducted in two main stages. In the first stage, a new A7G point mutation was introduced to the 3D structure of rhG-CSF by means of computational design. The mutant model, following energy minimization, displayed satisfactory stereochemistry (Molprobity clashscore 2.32) and maintained the native fold, suggesting that the mutation is structurally compatible. The second phase involved optimization of a high-yield expression system with the use of recombinant vector pET3a in E. coli BL21(DE3). Optimization of culture conditions showed that 4XYT medium with 0.5 mM IPTG and a 17-hour induction time resulted in the highest level of expression of the mutant protein. Such conditions led to a very high biomass yield of 96 grams of cells. The novelty of the present research work lies in the incorporation of the structure-guided single residue modification and process optimization in one step, resulting in structurally validated A7G rhG-CSF and process optimization to 5 L scale in complex medium.