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Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis
BACKGROUND: One of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies. Here a series of ligation independent clonin...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396618/ https://www.ncbi.nlm.nih.gov/pubmed/18485215 http://dx.doi.org/10.1186/1472-6750-8-51 |
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author | Qin, Huajun Hu, Jian Hua, Yuanzhi Challa, Shridhar V Cross, Timothy A Gao, Fei P |
author_facet | Qin, Huajun Hu, Jian Hua, Yuanzhi Challa, Shridhar V Cross, Timothy A Gao, Fei P |
author_sort | Qin, Huajun |
collection | PubMed |
description | BACKGROUND: One of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies. Here a series of ligation independent cloning based vectors were constructed to address this challenge. RESULTS: The feasibility of these vectors was tested with 41 putative membrane proteins from Mycobacterium tuberculosis. The efficiency for direct cloning of these target genes from PCR products was 95% (39/41). Over 40% of cloned genes were overexpressed in Escherichia coli BL21 (DE3)-RP codon plus strain in the first round of expression screening. For those proteins which showed no expression, three protein fusion partners were prepared and it was found that each of the target proteins could be overexpressed by at least one of these fusions, resulting in the overexpression of two thirds of the cloned genes. CONCLUSION: This expression platform features high throughput cloning, high flexibility for different constructs, and high efficiency for membrane protein overexpression, and is expected to be useful in membrane protein structural and functional studies. |
format | Text |
id | pubmed-2396618 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-23966182008-05-28 Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis Qin, Huajun Hu, Jian Hua, Yuanzhi Challa, Shridhar V Cross, Timothy A Gao, Fei P BMC Biotechnol Methodology Article BACKGROUND: One of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies. Here a series of ligation independent cloning based vectors were constructed to address this challenge. RESULTS: The feasibility of these vectors was tested with 41 putative membrane proteins from Mycobacterium tuberculosis. The efficiency for direct cloning of these target genes from PCR products was 95% (39/41). Over 40% of cloned genes were overexpressed in Escherichia coli BL21 (DE3)-RP codon plus strain in the first round of expression screening. For those proteins which showed no expression, three protein fusion partners were prepared and it was found that each of the target proteins could be overexpressed by at least one of these fusions, resulting in the overexpression of two thirds of the cloned genes. CONCLUSION: This expression platform features high throughput cloning, high flexibility for different constructs, and high efficiency for membrane protein overexpression, and is expected to be useful in membrane protein structural and functional studies. BioMed Central 2008-05-16 /pmc/articles/PMC2396618/ /pubmed/18485215 http://dx.doi.org/10.1186/1472-6750-8-51 Text en Copyright © 2008 Qin et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article Qin, Huajun Hu, Jian Hua, Yuanzhi Challa, Shridhar V Cross, Timothy A Gao, Fei P Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis |
title | Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis |
title_full | Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis |
title_fullStr | Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis |
title_full_unstemmed | Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis |
title_short | Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis |
title_sort | construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from mycobacterium tuberculosis |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396618/ https://www.ncbi.nlm.nih.gov/pubmed/18485215 http://dx.doi.org/10.1186/1472-6750-8-51 |
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