Expression and purification of human dystrophin Dp71 in Escherichia coli

Marco Gamboa-Melendez, Pablo Gómez, Mario Bermúdez De León, Miguel Rojas-Sierra, Bulmaro Cisneros

Research output: Contribution to journalArticle

Abstract

In recent years, along with the rapid development of genetic engineering technologies, a vast amount of valuable proteins have been expressed in Escherichia coli with high productivity. In order to produce large amounts of human dystrophin Dp71, a protein of unknown function, its coding sequence was cloned into the prokaryotic expression vector pMAL-c2X generating the gene fusion malE-Dp71. Screening of BL21, a protease-deficient strain, expressing the gene fusion malE-Dp71 was achieved by SDS-PAGE gel and Western blotting with an anti-Dp71 polyclonal antibody. The maltose-binding protein-Dp71 (MBP-Dp71) fusion protein, expressed in E. coli, was recovered into the soluble fraction of cell lysates using the sarkosyl detergent, and purified to homogeneity by affinity chromatography on amylose columns. The expression and purification procedures yielded approximately 98.5 μg of highly purified protein per 100 ml of bacterial culture. The expression system established in this study will provide the biological material needed for in vitro studies of Dp71.
Original languageEnglish
Pages (from-to)157-161
Number of pages5
JournalAnnals of Microbiology
Publication statusPublished - 11 Jul 2005
Externally publishedYes

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Dystrophin
Gene Fusion
Maltose-Binding Proteins
Escherichia coli
Amylose
Genetic Engineering
Affinity Chromatography
Detergents
Polyacrylamide Gel Electrophoresis
Proteins
Peptide Hydrolases
Western Blotting
Gels
Technology
Antibodies
apo-dystrophin 1
In Vitro Techniques
sarkosyl

Cite this

Gamboa-Melendez, M., Gómez, P., Bermúdez De León, M., Rojas-Sierra, M., & Cisneros, B. (2005). Expression and purification of human dystrophin Dp71 in Escherichia coli. Annals of Microbiology, 157-161.
Gamboa-Melendez, Marco ; Gómez, Pablo ; Bermúdez De León, Mario ; Rojas-Sierra, Miguel ; Cisneros, Bulmaro. / Expression and purification of human dystrophin Dp71 in Escherichia coli. In: Annals of Microbiology. 2005 ; pp. 157-161.
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Gamboa-Melendez, M, Gómez, P, Bermúdez De León, M, Rojas-Sierra, M & Cisneros, B 2005, 'Expression and purification of human dystrophin Dp71 in Escherichia coli', Annals of Microbiology, pp. 157-161.

Expression and purification of human dystrophin Dp71 in Escherichia coli. / Gamboa-Melendez, Marco; Gómez, Pablo; Bermúdez De León, Mario; Rojas-Sierra, Miguel; Cisneros, Bulmaro.

In: Annals of Microbiology, 11.07.2005, p. 157-161.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Expression and purification of human dystrophin Dp71 in Escherichia coli

AU - Gamboa-Melendez, Marco

AU - Gómez, Pablo

AU - Bermúdez De León, Mario

AU - Rojas-Sierra, Miguel

AU - Cisneros, Bulmaro

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AB - In recent years, along with the rapid development of genetic engineering technologies, a vast amount of valuable proteins have been expressed in Escherichia coli with high productivity. In order to produce large amounts of human dystrophin Dp71, a protein of unknown function, its coding sequence was cloned into the prokaryotic expression vector pMAL-c2X generating the gene fusion malE-Dp71. Screening of BL21, a protease-deficient strain, expressing the gene fusion malE-Dp71 was achieved by SDS-PAGE gel and Western blotting with an anti-Dp71 polyclonal antibody. The maltose-binding protein-Dp71 (MBP-Dp71) fusion protein, expressed in E. coli, was recovered into the soluble fraction of cell lysates using the sarkosyl detergent, and purified to homogeneity by affinity chromatography on amylose columns. The expression and purification procedures yielded approximately 98.5 μg of highly purified protein per 100 ml of bacterial culture. The expression system established in this study will provide the biological material needed for in vitro studies of Dp71.

M3 - Article

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Gamboa-Melendez M, Gómez P, Bermúdez De León M, Rojas-Sierra M, Cisneros B. Expression and purification of human dystrophin Dp71 in Escherichia coli. Annals of Microbiology. 2005 Jul 11;157-161.