Prokaryotic epression, prurification and identification of PTD-FNK protien

ObjectiveThe mutant PTD-FNK protein of Bcl-xL protein was expressed in prokaryotic cell , in order to reveal anti-apoptosis mechanism and accelerate its application in livestock semen cryopreservation. MethodBased on nu-cleotide sequence of FNKT protein, two primers were designed for site-specific mutagenesis, two mutated fragments were amplified by PCR using pET-28a(+)-PTD-Bcl-xL plasmid as template. The extracted PCR products were digested by Dpn l to construct recombinant plasmid, and transformed into Escherichia coli DH5α. Then fusion protein was expressed by IPTG induction, and its optimum induction temperature was studied. Furthermore, the expressed fusion protein was pu-rified by NI-NTA affinity chromatography and identified by SDS-PAGE and Western bloting. ResultThe PCR products of two site-specific mutagenesis were 462 and 6093 bp in length. After restriction enzyme digestion , recombination and transformation, pET-28a(+)-PTD-FNK plasmid was successfully constructed. The fusion protein was well expressed by inducing its gene with IPTG at 30 ℃ for 7 h. SDS-PAGE and Western bloting results showed the purified protein was PTD-FNK protein. ConclusionThe soluble PTD-FNK fusion protein is expressed successfully by regulating induction temperature. Furthermore, it has a lots of advantages, such as shorter induction time, without refolding, easier protein purification, accelerating practical application of PTD-FNK protein in livestock semen cryopreservation.