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Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering
Cited 2 time in
Web of Science
Cited 1 time in Scopus
- Authors
- Issue Date
- 2020-07
- Publisher
- American Chemical Society
- Citation
- ACS Synthetic Biology, Vol.9 No.7, pp.1591-1598
- 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 subdoning 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 doning 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.
- ISSN
- 2161-5063
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