Prokaryotic expression and purification of PTD-SH2 fusion protein

AIM: To construct the recombinant expression vector containing protein transduction domain (PTD) and Src homology 2 (SH2) fusion gene and express PTD-SH2 fusion protein in E. coli and purify the fusion protein. METHODS: A 297 bp of human SH2 gene fragment was amplified by PCR and subcloned into pET-16b vector downstream of the PTD fragment, an E.coli expression vector, to construct a recombinant plasmid pET-16b-PTD-SH2. The plasmid was transformed into E.coli BL21 (DE3) and induced to express fusion protein PTD-SH2 with IPTG. The expression of PTD-SH2 was detected by SDS-PAGE and Western blot. The expressed protein was purified by Ni-NTA affinity chromatographic column. RESULTS: SH2 was identical to what reported by GenBank. A novel protein with expected molecular mass (about Mr 15×103) was expressed under the induction with IPTG. The expressed product showed good reactivity to anti-His tag antibody, and was mostly in the form of inclusion bodies. The expressed proteins could be purified via Ni-NTA affinity chromatography in denatured condition. CONCLUSION: Our successful construction of recombinant express vector pET-16b-PTD-SH2 and efficient expression of PTD-SH2 fusion protein in E. coli and purification of interest protein lay a basis for further study on SH2 functions.