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Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories
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 for...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037224/ https://www.ncbi.nlm.nih.gov/pubmed/31973001 http://dx.doi.org/10.3390/ijms21030688 |
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author | Sohail, Anil A. Gaikwad, Madhuri Khadka, Prakash Saaranen, Mirva J. Ruddock, Lloyd W. |
author_facet | Sohail, Anil A. Gaikwad, Madhuri Khadka, Prakash Saaranen, Mirva J. Ruddock, Lloyd W. |
author_sort | Sohail, Anil A. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7037224 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-70372242020-03-11 Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories Sohail, Anil A. Gaikwad, Madhuri Khadka, Prakash Saaranen, Mirva J. Ruddock, Lloyd W. Int J Mol Sci Article 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. MDPI 2020-01-21 /pmc/articles/PMC7037224/ /pubmed/31973001 http://dx.doi.org/10.3390/ijms21030688 Text en © 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/). |
spellingShingle | Article Sohail, Anil A. Gaikwad, Madhuri Khadka, Prakash Saaranen, Mirva J. Ruddock, Lloyd W. Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories |
title | Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories |
title_full | Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories |
title_fullStr | Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories |
title_full_unstemmed | Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories |
title_short | Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories |
title_sort | production of extracellular matrix proteins in the cytoplasm of e. coli: making giants in tiny factories |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037224/ https://www.ncbi.nlm.nih.gov/pubmed/31973001 http://dx.doi.org/10.3390/ijms21030688 |
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