High-level expression and purification of human γD-crystallin in Escherichia coli

Abstract Human γD-crystallin (HγDC), a 173 amino acid protein, is a primary protein component of the human eye lens. It is composed of two highly homologous β-sheet domains and is associated with the development of juvenile and adult-onset cataracts. In the present study, we describe the expression, purification, and characterization of HγDC. We optimized recombinant protein expression in an Escherichia coli expression system by investigating factors such as the type of promoter, E. coli strain, culture temperature, isopropyl β- d -thiolgalactorpyranoside (IPTG) concentration, optical density of induction (OD 600 nm ), and duration of IPTG induction. We then successfully purified recombinant HγDC coupled to a six-histidine tag (6×His-HγDC). Our results revealed that the optimal system for 6×His-HγDC protein expression was culture of E. coli strain BL21(DE3) bearing the pEHisHγDC plasmid at 30 °C and induction with 0.5 mM IPTG once the culture reached an optical density of 2.5 for a period of 8 h. Circular dichroism spectroscopy and fluorescence spectroscopy demonstrated that the structural integrity of the purified 6×His-HγDC protein was identical to that of its native counterpart. Our further investigation also revealed that almost no structural difference was detected between HγDC with and without His-tags. The expression and purification procedure was optimized and the resultant final yield (∼23.3 mg/100 mL of culture medium; i.e., ∼23.3 mg protein produced in 100 mL of culture medium) was significantly (∼5–10-fold) higher than those from previous reports.