Cloning, Prokaryotic Expression Of Capra Hircus Klf4,and Purification Of HIS-KLF4Fusion Protein

[Objective]KLF4(Krueppel-like factor4) is a kind of transcription factor to matain pluripotency of stem cells, which plays an important role in reprogramming adult cells to induced pluripotent stem cells(iPS cells). The present paper was to clone K1f4gene of Capra Hircus, to contruct its prokaryotic expression vector, and further to induce expression of His-Klf4fusion protein in E.coli BL21(DE3), which would be purified finally.[Methods] The total RNA was extracted from primodial genital ridges of fetal Capra Hircus at age of six weeks, and by means of RT-PCR, the coding sequences of DAZL gene was amplified, and recombinant plasmid pMD18-T-klf4was constructed by TA cloning. After restriction endonuclease digestion and sequencing, k1f4cDNA was subcloned to the pET30a vector to construct recombinant plasmid pET30a-klf4. Identified again by restriction endonuclease digestion, the recombinant plasmid was transformed into in E.coli BL21(DE3), and His-Klf4 fusion protein was expressed by induction of IPTG, which was verified by SDS-PAGE analysis and Western blotting assay. Finally, the recombinant His-klf4fusion protein was purified by metal Nickel-chelating affinity chromatography.[Results](1) The klf4gene was successfully cloned from the promodial genital ridges of fetal Capra Hircus; Sequence analysis showed that The open reading frame(ORF) of Capra Hircus Klf4is composed of1434nucleutide acids, coding478amino acids; A comparison of sequence similarity of Capra Hircus Klf4homologue with other vertebrate Klf4homologues indicated that Capra Hircus shared the highest homology(98.5%) with Ovis Aries.(2) Signal peptide prediction by SignalP4.0online showed that there was no signal peptide in the coding proteins of Capra Hircus Klf4.(3) SDS-PAGE and Western blotting assay showed that the recombinant plasmid was expressed in E.coli BL21(DE3); His-Klf4fusion protein was purified under denature condition.[Conclusion] The Klf4of Capra Hircus gene was successfully cloned, and His-Klf4fusion protein was obtained through prokaryotic expression technology, which will contribute to prepare polyclonal anti-Klf4antibody, and further to reveal self-renew mechanisms of Capra Hircus iPS cells.