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

Jihyeon Yu, Eunju Cho, Yeon Gil Choi, You Kyeong Jeong, Yongwoo Na, Jong Seo Kim, Sung Rae Cho, Jae Sung Woo, Sangsu Bae, Jae Sung Woo

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

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.

Original languageEnglish
Pages (from-to)1591-1598
Number of pages8
JournalACS Synthetic Biology
Volume9
Issue number7
DOIs
Publication statusPublished - 2020 Jul 17

Keywords

  • CRISPR-Cas system
  • Reelin
  • genome engineering
  • molecular cloning
  • protein purification

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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