Expression of human NGFβ gene segment encoding mature peptide in E.coli and determination of its bioactivity
Ma Wei1, Liu Miao1, Yang Guangxiao2, Wang Quanying2 (1. Department of Orthopedics, First Hospital of Xi'an Jiaotong University, Xi'an 710061; 2. Xi'an Huaguang Biological Engineering, INC, Xi'an 710025, China)
Objective To express the hNGFβ gene segment encoding mature peptide (hNGFβ ) in E.coli and determine its bioactivity. Methods The resulting gene of hNGFβ was subclonedinto the hNGFβ site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E.coli DH 5α. The proteins of hNGFβ were expressed by temperature induction. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the expressed hNGFβ tests of neurite growth of dorsal root knot of chicken embryo and tests of Brdu incorporation into PC12 cells was a biologically active protein. Results The recombinant plasmid pBV220/NGFβ was successfully constructed. The NGFβ was inserted to pBV220 plasma, a prokaryotic expression vector. Expression of NGFβ in E.coli was induced by raising temperature to 42℃. SDS-PAGE electrophoresis showed that NGFβ protein existed in inclusion. The solubility protein of NGFβ was obtained through purification of inclusion by centrifugation and technique of protein repatriation. Recombinant NGFβ protein was purified by affinity chromatography of heparin SepharoseCL-6B. The purity of NGFβ was higher than 90% and yield of NGFβ was 1.8～2.0mg/L expressing bacteria. The bioactivity of NGFβ expressing prokaryotic cell was 1×10 5BU/g according to rule concerning examination of biological products in China. Conclusion The hNGFβgene with bioactivity can be expressed in E.coli.