The expression and purification of a new complement inhibitor complement receptor of the immunoglobulin superfamily

Abstract

Objective To construct the prokaryotic expression plasmid pSmartI-SUMO-complement receptor of the immunoglobulin superfamily (CRIg) of the CRIg, next express and purify it by E. coli expression system, finally detect the activity of the expressed product. Methods Total RNA was extracted from human liver cell HHMa, and the gene sequence encoding the extracellular domain of human CRIg was cloned onto the pSmart-I vector by reverse transcriptase-polymerase chain reaction (RT-PCR) and Seamless cloning. Positive colonies were selected for plasmid extraction and sequencing, and the expressed products were detected by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. Finally, the activity was detected by alternative pathway hemolysis assay. The experiment was repeated three times and averaged to plot the hemolysis curve. Results The PCR product amplified from human CRIg was 819 bp in length. The sequencing result was aligned with the extracellular segment of human CRIg. After the plasmid was transformed into Rosetta strain, it was found by IPTG that CRIg could be stably expressed in the supernatant. Hemolysis experiments demonstrated that a 25 μm protein concentration could almost completely inhibit hemolysis. Conclusion CRIg can achieve soluble expression in prokaryotic expression system. Key words: Complement receptor of the immunoglobulin superfamily gene; Complement; Immune inhibitor; Seamless cloning; Prokaryotic expression plasmid