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Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering
journal contribution
posted on 2020-07-02, 11:33 authored by Jihyeon Yu, Eunju Cho, Yeon-Gil Choi, You Kyeong Jeong, Yongwoo Na, Jong-Seo Kim, Sung-Rae Cho, Jae-Sung Woo, Sangsu BaeThe
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.