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Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering

Authors
 Jihyeon Yu  ;  Eunju Cho  ;  Yeon-Gil Choi  ;  You Kyeong Jeong  ;  Yongwoo Na  ;  Jong-Seo Kim  ;  Sung-Rae Cho  ;  Jae-Sung Woo  ;  Sangsu Bae 
Citation
 ACS SYNTHETIC BIOLOGY, Vol.9(7) : 1591-1598, 2020-06 
Journal Title
 ACS SYNTHETIC BIOLOGY 
Issue Date
2020-06
Keywords
CRISPR-Cas system ; Reelin ; genome engineering ; molecular cloning ; protein purification
Abstract
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.
Full Text
https://pubs.acs.org/doi/10.1021/acssynbio.0c00090
DOI
10.1021/acssynbio.0c00090
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Rehabilitation Medicine (재활의학교실) > 1. Journal Papers
Yonsei Authors
Cho, Sung-Rae(조성래) ORCID logo https://orcid.org/0000-0003-1429-2684
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/179429
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