Expression and purification of recombinant human α 1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity

Abstract

Human α 1-proteinase inhibitor (α 1-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant α 1-PI has great potential in therapeutic applications. We report here the expression of a synthetic α 1-PI gene and its variants in Escherichia coli. Modified α 1-PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant α 1-PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant α 1-PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7–9 mg l −1 of bacterial culture (∼5.2 g wet cell mass). E. coli expressed recombinant α 1-PI showed specific anti-elastase activity and appeared as a single band of ∼45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant α 1-PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified α 1-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active α 1-PI and its variants in E. coli for further therapeutic applications.