| Expression of Recombinant Chinese Bovine Enterokinase Catalytic Subunit in P. pastoris and Its Purification and Characterization |
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| 作者姓名： | Fang L Sun QM Hua ZC |
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| 作者单位： | Lei FANG，Qi-Ming SUN，and Zi-Chun HUA*The State Key Laboratory of Pharmaceutical Biotechnology，Nanjing University，Nanjing 210093，China |
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| 基金项目： | The study was supported by a grant from the Teaching and Research
Award Program for the Outstanding Young Teachers in Higher Education
Institutions of Ministry of Education of China |
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| 摘 要： | EK (enterokinase) is a serine proteinase which consistsof a heavy chain and a light chain linked by a disulfidebond. The light chain of EK contains a chymotrypsin-likeserine proteinase domain sufficient for the normal recog-nition and cleavage of EK subst…
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| 关 键 词： | 重组体 肠激酶 催化亚单位 中国牛 融合蛋白 分泌表达 |
Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization |
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| Fang L,Sun QM,Hua ZC.Expression of Recombinant Chinese Bovine Enterokinase Catalytic Subunit in P. pastoris and Its Purification and Characterization[J].Acta Biochimica et Biophysica Sinica,2004,36(7):513-517. |
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| Authors: | Fang Lei Sun Qi-Ming Hua Zi-Chun |
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| Institution: | The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China. |
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| Abstract: | Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins. cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EKL) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of alpha-mating factor from Saccharomyces cerevisiae to get the alpha-MF signal-EKL-His6 encoding gene by PCR. Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris. Secreted expression of recombinant EKL-His6 was attained by methanol induction and its molecular weight is 43 kD. Because of the existence of His6-tag, EKL-His6 was easily purified from P. pastoris fermentation supernatant by using Ni2+ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture. This purified EKL-His6 demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site. |
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| Keywords: | recombinant enterokinase secreted expression Ni2 affinity chromatography fusion protein cleavage |
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