Human alpha(1)-proteinase inhibitor (alpha(1)-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant alpha(1)-PI has great potential in therapeutic applications. We report here the expression of a synthetic alpha(1)-PI gene and its variants in Escherichia coli. Modified alpha(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 alpha(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 alpha(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 (approximately 5.2 g wet cell mass). E. coli expressed recombinant alpha(1)-PI showed specific anti-elastase activity and appeared as a single band of approximately 45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant alpha(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 alpha(1)-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active alpha(1)-PI and its variants in E. coli for further therapeutic applications.
Submitter: Shu-Ching Ou
Submission Date: Nov. 29, 2018, 3:57 p.m.
|Number of data points||42|
|Assays/Quantities/Protocols||Experimental Assay: Biologically Active Content ; Experimental Assay: Recombinant protein content ; Experimental Assay: Total soluble protein content ; Derived Quantity: SD of Specific Activity ; Derived Quantity: Specific Activity ; Derived Quantity: SD of Biologically Active Content ; Derived Quantity: SD of Recombinant protein content|
|Libraries||Variants for Alpha-1-proteinase inhibitor_SLTP ; Variants for Alpha-1-proteinase inhibitor|