Cloning and Prokaryotic Expression of Capra hircus Klf4，and Purification of His-Klf4 Fusion Protein

Abstract

Abstract: The paper was to clone Klf4 gene of Capra hircus, and construct recombinant plasmid pET30a-klf4, and finally to prepare the purified His-klf4 fusion protein by means of prokaryotic expression techniques . Total RNA extracted from the genital ridge of fetal Capra hircus, Klf4 cDNA was amplified by RT-PCR and the plasmid pMD18-T-Klf4 was constructed by TA cloning. After restriction endonuclease digestion and sequencing analysis, Klf4 cDNA was subcloned to pET-30a vector to obtain a recombinant plasmid pET30a-Klf4. Transformed into E. coli BL21, the recombinant plasmid pET30a-Klf4 was induced to express by IPTG, which was verified by SDS-PAGE and Western blotting analysis. Finally, the His-Klf4 fusion protein was purified via metal Nickel-chelating affinity chromatography. The results showed that ：（1）the Klf4 gene was successfully cloned from promodial genital ridges of fetal Capra hircus; the open reading frame（ORF）of Capra hircus Klf4 is composed of 1434 nucleutide acids, coding 478 amino acids; comparison of sequence similarity of Capra hircus Klf4 homologue with other vertebrate Klf4 homologues indicated that Capra hircus shared the highest homology（98.5%）with Ovis aries.（2）Signal peptide prediction by SignalP 4.0 online 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, and His-Klf4 fusion protein was purified under denature condition.

Key words: Klf4, Molecular cloning, Prokaryotic expression, Protein purification, Capra hircus

Xin Guiyu, Wang Lixia, Ye Xinhui, Shen Kuikui, Fu Xinhui, Li Gonghe, Zhang Ming, Lu Shengsheng, Lu Kehuan, Zheng Xibang. Cloning and Prokaryotic Expression of Capra hircus Klf4，and Purification of His-Klf4 Fusion Protein[J]. Biotechnology Bulletin, 2013, 0(2): 80-85.

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