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.
|Number of pages||8|
|Journal||ACS Synthetic Biology|
|Publication status||Published - 2020 Jul 17|
Bibliographical noteFunding Information:
This research was supported by grants from the National Research Foundation of Korea (NRF) (no. 2018R1C1B6004447 to J.-S.W., no. 2018M3A9H3022412 to S.B.) and by Next Generation BioGreen 21 Program (PJ01319301), Korea Healthcare Technology R&D Project (HI16C1012), the Technology Innovation Program (no. 20000158) to S.B. The authors thank Medical Illustration & Design, part of the Medial Research Support Services of Yonsei University College of Medicine, for a cover artistic support related to this work.
Copyright © 2020 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)