Prokaryotic expression and purification of human endogenous Borna-like N element-1

Objective To express human endogenous Borna-like N element-1(EBLN-1)in prokaryotic cells,and purify and identify the expressed product. Methods EBLN-1 gene was amplified by PCR using synthetic EBLN-1 gene as a template and inserted into vector pET-41a. The constructed recombinant plasmid pET-41a-EBLN-1 was transformed to E.coli BL21(DE3)and Rosetta,and induced with IPTG at 18 and 37 ℃,respectively. The expressed protein was refolded by dilution of inclusion body,purified by His affinity chromatography,and identified by SDS-PAGE and Western blot. Results Colony PCR and sequencing proved that recombinant plasmid pET-41a-EBLN-1 was constructed correctly. Recombinant EBLN-1 protein,with a relative molecular mass of about 48 000,was expressed in a form of inclusion body,of which the expression level in E.coli Rosetta reached a maximum of 30% of total somatic protein after induction at 37 ℃. The purified protein reached a purity of 90% and showed specific binding to HRP-labeled His probe. Conclusion Recombinant EBLN-1 protein was successfully expressed and purified,which laid a foundation of further study on physiological role of EBLN-1 in humans as well as its relationship to mental diseases.