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Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography

Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich rec...

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Main Authors: Oyeleye, Ayokunmi Omolola, Mohd Yusoff, Siti Faridah, Abd Rahim, Izzah Nadiah, Thean, Adam Chor Leow, Saidi, Noor Baity, Mohd Yahaya, Normi
Format: Article
Language:English
Published: Public Library of Science 2020
Online Access:http://psasir.upm.edu.my/id/eprint/88662/1/ABSTRACT.pdf
http://psasir.upm.edu.my/id/eprint/88662/
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241074
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spelling my.upm.eprints.886622021-12-15T00:07:41Z http://psasir.upm.edu.my/id/eprint/88662/ Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography Oyeleye, Ayokunmi Omolola Mohd Yusoff, Siti Faridah Abd Rahim, Izzah Nadiah Thean, Adam Chor Leow Saidi, Noor Baity Mohd Yahaya, Normi Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies. Public Library of Science 2020 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/88662/1/ABSTRACT.pdf Oyeleye, Ayokunmi Omolola and Mohd Yusoff, Siti Faridah and Abd Rahim, Izzah Nadiah and Thean, Adam Chor Leow and Saidi, Noor Baity and Mohd Yahaya, Normi (2020) Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography. PLoS One, 15 (10). art. no. 0241074. pp. 1-21. ISSN 1932-6203 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241074 10.1371/journal.pone.0241074
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.
format Article
author Oyeleye, Ayokunmi Omolola
Mohd Yusoff, Siti Faridah
Abd Rahim, Izzah Nadiah
Thean, Adam Chor Leow
Saidi, Noor Baity
Mohd Yahaya, Normi
spellingShingle Oyeleye, Ayokunmi Omolola
Mohd Yusoff, Siti Faridah
Abd Rahim, Izzah Nadiah
Thean, Adam Chor Leow
Saidi, Noor Baity
Mohd Yahaya, Normi
Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
author_facet Oyeleye, Ayokunmi Omolola
Mohd Yusoff, Siti Faridah
Abd Rahim, Izzah Nadiah
Thean, Adam Chor Leow
Saidi, Noor Baity
Mohd Yahaya, Normi
author_sort Oyeleye, Ayokunmi Omolola
title Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
title_short Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
title_full Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
title_fullStr Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
title_full_unstemmed Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
title_sort effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from streptomyces griseus by reverse dilution and affinity chromatography
publisher Public Library of Science
publishDate 2020
url http://psasir.upm.edu.my/id/eprint/88662/1/ABSTRACT.pdf
http://psasir.upm.edu.my/id/eprint/88662/
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241074
_version_ 1720438539557011456
score 13.211869

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