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Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone
The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8541268/ https://www.ncbi.nlm.nih.gov/pubmed/34684787 http://dx.doi.org/10.3390/molecules26206207 |
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author | Jiang, Dai-Lin Yao, Chao-Ling Hu, Nien-Jen Liu, Yung-Chuan |
author_facet | Jiang, Dai-Lin Yao, Chao-Ling Hu, Nien-Jen Liu, Yung-Chuan |
author_sort | Jiang, Dai-Lin |
collection | PubMed |
description | The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application. |
format | Online Article Text |
id | pubmed-8541268 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-85412682021-10-24 Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone Jiang, Dai-Lin Yao, Chao-Ling Hu, Nien-Jen Liu, Yung-Chuan Molecules Article The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application. MDPI 2021-10-14 /pmc/articles/PMC8541268/ /pubmed/34684787 http://dx.doi.org/10.3390/molecules26206207 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Jiang, Dai-Lin Yao, Chao-Ling Hu, Nien-Jen Liu, Yung-Chuan Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone |
title | Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone |
title_full | Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone |
title_fullStr | Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone |
title_full_unstemmed | Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone |
title_short | Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone |
title_sort | construction of a tandem repeat peptide sequence with pepsin cutting sites to produce recombinant α-melanocyte-stimulating hormone |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8541268/ https://www.ncbi.nlm.nih.gov/pubmed/34684787 http://dx.doi.org/10.3390/molecules26206207 |
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