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利用酵母表达系统从藏绵羊乳腺中克隆、表达和纯化乳铁蛋白

Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system.

作者信息

Li Jianbo, Zhu Wuzheng, Luo Meirong, Ren Honghui, Tang Lu, Liao Honghai, Wang Yong

机构信息

Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, PR China; College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.

College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China.

出版信息

Protein Expr Purif. 2015 May;109:35-9. doi: 10.1016/j.pep.2015.01.008. Epub 2015 Jan 31.

Abstract

This paper reports the successful expression of a lactoferrin gene-obtained from the mammary gland tissue of Tibetan sheep-in the yeast Pichia pastoris GS115 using pPICZαA as the recombinant plasmid and α-factor signal sequence for secretion. The recombinant lactoferrin was purified by ammonium sulfate precipitation, ion-exchange column chromatography and gel-filtration chromatography, and it had a molecular mass of 76kDa. We obtained an expression yield of >60mgL(-1) and specific activity of 2533.33Umg(-1). The antimicrobial activities and iron-binding behaviors of recombinant lactoferrin indicated that it was correctly folded and functional. Additionally, recombinant lactoferrin inhibited the growth of Escherichia coli JM109 and Staphylococcus aureus. These findings indicate that recombinant lactoferrin is a potential antibiotic for use on humans. This study also demonstrates the successful expression of recombinant lactoferrin using the eukaryotic host organism P. pastoris, paving the way for protein engineering using this gene.

摘要

本文报道了以pPICZαA为重组质粒,利用α-因子信号序列进行分泌,成功地在毕赤酵母GS115中表达了从藏绵羊乳腺组织获得的乳铁蛋白基因。重组乳铁蛋白通过硫酸铵沉淀、离子交换柱色谱和凝胶过滤色谱进行纯化,其分子量为76kDa。我们获得了>60mgL(-1)的表达产量和2533.33Umg(-1)的比活性。重组乳铁蛋白的抗菌活性和铁结合行为表明其折叠正确且具有功能。此外,重组乳铁蛋白抑制了大肠杆菌JM109和金黄色葡萄球菌的生长。这些发现表明重组乳铁蛋白是一种潜在的用于人类的抗生素。本研究还证明了利用真核宿主生物巴斯德毕赤酵母成功表达重组乳铁蛋白,为利用该基因进行蛋白质工程铺平了道路。

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