Expression,purification of recombinant BDNF protein in E.coli. and its functional characterization

Objective To construct the prokaryotic expression vector for brain-derived neurotrophic factor(BDNF)gene and express it in E.coli,so as to obtain the recombinant BDNF protein with high yield,low cost,high purity and high biological activity.Methods The cDNA fragment from maturation region of BDNF was obtained by standard PCR method with a full-length human BDNF cDNA as template,and was inserted into plasmid pET-30a(+)by means of gene re-arrangement.The recombinant plasmid pET-BDNF was identified by restriction endonuclease analysis and DNA sequencing.The cloned pET-BDNF plasmid was transformed into E.coli host strain,BL21(DE3)-LysS.Following induction by IPTG,the recombinant protein was expressed and then purified by Ni-NAT affinity chromatography under denaturing conditions.The interest protein was viewed by SDS-PAGE,further characterized by western blot.The proliferation of PC12 cells was evaluated by MTT assay.Results The human BDNF cDNA was amplified and cloned into prokaryotic expression vector.The restriction endonuclease analysis and DNA sequencing proved that the plasmid pET-BDNF was obtained.The recombinant protein was expressed in E.coli system.After Coomassie brilliant blue staining,the recombinant protein showed an exclusive band;western blot analysis with anti-BDNF antibody and anti-his antibody supported that the expressed protein was recombinant BDNF.The recombinant human BDNF protein could enhance the proliferation of PC12 cells.Conclusion The prokaryotic expression vector for human BDNF is successfully constructed.The recombinant human BDNF protein can be expressed and purified in E.coli.and has a good biological activity.