Prokaryotic expression,purification and antiserum preparation of X-linked inhibitor of apoptosis protein

Objective:To purify human XIAP recombinant protein expressed in the E.coli,and to generate its antiserum. Methods:Vector pET30a(+)/XIAP was constructed and transformed into E.coli BL21(DE3).The recombinant protein was produced after the induction of 0.1mmol/L isopropyl-1-thio-D-galactopyranside(IPTG).The cell pellet was lysed by the combined use of sonication and lysozyme.The pellet of inclusion bodies was washed respectively with binding buffer which containing 2 mol/L,4 mol/L urea and dissolved with binding buffer containing 8 mol/L urea.The recombinant protein sample was obtained by eluted Ni-NTA column with elution buffer which containing 8 mol/L urea.SDS-PAGE gel electrophoresis was carried out to analyze the specificity of recombinant protein.BALB/c mice were immunized by the purified protein,and ELISA was employed to detect the titer of the antiserum. Results:Human XIAP recombinant protein prokaryotic expression vector was successfully constructed by seamless cloning method.The recombinant protein was expressed as inclusion bodies and its molecular weight was about 54 kD,consistent with the expecting value.Through affinity chromatography recombinant protein was purified and the purity coefficient was up to 90%.The antiserum had high titer(1∶50 000). Conclusion:High purified human XIAP recombinant protein and the antiserum may facilitate further functional study in the human XIAP protein and development of diagnostic reagents.