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

Human gamma D-crystallin (H gamma DC), a 173 amino acid protein, is a primary protein component of the human eye lens. It is composed of two highly homologous beta-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 H gamma DC. 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 beta-D-thiolgalactorpyranoside (IPTG) concentration, optical density of induction (OD600 nm). and duration of IPTG induction. We then successfully purified recombinant H gamma DC coupled to a six-histidine tag (6xHis-H gamma DC). Our results revealed that the optimal system for 6xHis-H gamma DC protein expression was culture of E. coli strain BL21(DE3) bearing the pEHisH gamma DC plasmid at 30 degrees 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 6xHis-H gamma DC protein was identical to that of its native counterpart. Our further investigation also revealed that almost no structural difference was detected between H gamma DC with and without His-tags. The expression and purification procedure was optimized and the resultant final yield (similar to 23.3 mg/100 mL of culture medium; i.e., similar to 23.3 mg protein produced in 100 mL of culture medium) was significantly (similar to 5-10-fold) higher than those from previous reports. (C) 2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.