Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering.,ACS Synthetic Biology

当前位置： X-MOL 学术 › ACS Synth. Biol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-06-25 , DOI: 10.1021/acssynbio.0c00090
Jihyeon Yu _{1,

2} , Eunju Cho _{3,

4} , Yeon-Gil Choi ₅ , You Kyeong Jeong _{1,

2} , Yongwoo Na _{6,

7} , Jong-Seo Kim _{6,

7} , Sung-Rae Cho _{3,

4,

8} , Jae-Sung Woo ₅ , Sangsu Bae _{1,

2}

Affiliation

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.

中文翻译：

通过直接基因组工程纯化从内源基因座过表达的完整人类蛋白质。

人类蛋白质的过量生产和纯化是基础研究和医学研究的必要条件。尽管许多重组人蛋白已被纯化，但目前的蛋白生产方法仍存在一些局限性；重组蛋白经常被截短、无法正确折叠和/或缺乏适当的翻译后修饰。此外，此类方法需要将靶基因亚克隆到相关质粒中，这对于具有重复结构域的长蛋白质来说可能很困难。在这里，我们设计了一种新的目标蛋白生产方法，通过在内源性人类基因前面引入用于过表达的强启动子和用于纯化的表位标签，从某种意义上说，直接在人类基因组中进行分子克隆，这不需要克隆目标基因。作为概念证明，我们成功纯化了完整的人类 Reelin 蛋白，该蛋白很长（3460 个氨基酸）且包含重复结构域，并证实其具有生物学功能。

更新日期：2020-07-17

点击分享查看原文

点击收藏

公开下载

阅读更多本刊最新论文本刊介绍/投稿指南11