University of Oulu

Sohail, A.A.; Gaikwad, M.; Khadka, P.; Saaranen, M.J.; Ruddock, L.W. Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories. Int. J. Mol. Sci. 2020, 21, 688. https://doi.org/10.3390/ijms21030688

Production of extracellular matrix proteins in the cytoplasm of E. coli : making giants in tiny factories

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Author: Sohail, Anil A.1; Gaikwad, Madhuri1; Khadka, Prakash1;
Organizations: 1Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, Finland
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 1.7 MB)
Persistent link: http://urn.fi/urn:nbn:fi-fe2020050825731
Language: English
Published: Multidisciplinary Digital Publishing Institute, 2020
Publish Date: 2020-05-08
Description:

Abstract

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies.

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Series: International journal of molecular sciences
ISSN: 1661-6596
ISSN-E: 1422-0067
ISSN-L: 1661-6596
Volume: 21
Issue: 3
Article number: 688
DOI: 10.3390/ijms21030688
OADOI: https://oadoi.org/10.3390/ijms21030688
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Subjects:
Funding: This work was supported by the Academy of Finland (grant number 272573) and Biocenter Oulu.
Academy of Finland Grant Number: 272573
Detailed Information: 272573 (Academy of Finland Funding decision)
Copyright information: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
  https://creativecommons.org/licenses/by/4.0/