Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243

Introduction: Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease endemic in Southeast Asia and northern Australia. Cases have been reported in Pahang, Johor Bahru and Kedah. The disease is difficult to combat as B. pseudomallei has shown resistance to various anti...

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Main Authors:	Mohamed Rehan, Aisyah, Ujang, Hanisah, Drahaman, Siti Marhamah, Mat Akhir, Nor Azurah, Muhamad Bunnori, Noraslinda, Raih, Mohd Firdaus
Format:	Conference or Workshop Item
Language:	English
Published:	2017
Subjects:	QR Microbiology
Online Access:	http://irep.iium.edu.my/57962/ http://irep.iium.edu.my/57962/19/57962.pdf

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spelling	iium-579622017-08-22T03:55:36Z http://irep.iium.edu.my/57962/ Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243 Mohamed Rehan, Aisyah Ujang, Hanisah Drahaman, Siti Marhamah Mat Akhir, Nor Azurah Muhamad Bunnori, Noraslinda Raih, Mohd Firdaus QR Microbiology Introduction: Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease endemic in Southeast Asia and northern Australia. Cases have been reported in Pahang, Johor Bahru and Kedah. The disease is difficult to combat as B. pseudomallei has shown resistance to various antibiotics and much is still not understood about its pathogenicity. It is suggested that investigating the bacterium's hypothetical proteins may provide potential new targets for the development of antimicrobials. The gene of interest in this study, BPSL2774, encoding BPSL2774 hypothetical protein, is a target gene that has been predicted as essential using transposon-directed insertion site sequencing technique (TraDIS). We aim to express and purify soluble GST-tagged BPSL2774 protein at sufficient concentration for future functional assays. Materials and method: The BPSL2774 gene has previously been amplified from genomic DNA of B. pseudomallei K96243 and cloned into pDEST15 (GST-tag) plasmid vector. In this work, the clone was transformed into E. coli BL21(DE3) expression strain cells for up-scaled protein preparations in 0.5 L and 1 L cultures. The auto-induction method was adopted for protein expression. GST-tag affinity chromatography was performed for protein purification and the fractions obtained were analyzed using SDS-PAGE. Results: The target protein was successfully expressed in soluble form and its highest concentration from a 0.8 mL elution fraction was at 1.38 mg/mL. Mass spectrometry analysis of 60 kDa coomassie-stained gel band cut confirmed the presence of the soluble expressed target protein, co-purified with E. coli chaperonin proteins, possibly due to their interaction with the target protein. Higher purity can be achieved through further purification steps following initial GST-tag affinity chromatography. Conclusion: The purified protein was at an acceptable purity and at sufficient concentration for use as samples in a glycosyltransferase bioluminescence assay in the near future. 2017 Conference or Workshop Item NonPeerReviewed application/pdf en http://irep.iium.edu.my/57962/19/57962.pdf Mohamed Rehan, Aisyah and Ujang, Hanisah and Drahaman, Siti Marhamah and Mat Akhir, Nor Azurah and Muhamad Bunnori, Noraslinda and Raih, Mohd Firdaus (2017) Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243. In: Medical Research Symposium 2017, 10th August 2017, Kulliyyah of Medicine, International Islamic University Malaysia, Indera Mahkota Campus, Jalan Sultan Ahmad Shah, Kuantan, Pahang Darul Makmur, Malaysia. (Unpublished)
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
language	English
topic	QR Microbiology
spellingShingle	QR Microbiology Mohamed Rehan, Aisyah Ujang, Hanisah Drahaman, Siti Marhamah Mat Akhir, Nor Azurah Muhamad Bunnori, Noraslinda Raih, Mohd Firdaus Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243
description	Introduction: Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease endemic in Southeast Asia and northern Australia. Cases have been reported in Pahang, Johor Bahru and Kedah. The disease is difficult to combat as B. pseudomallei has shown resistance to various antibiotics and much is still not understood about its pathogenicity. It is suggested that investigating the bacterium's hypothetical proteins may provide potential new targets for the development of antimicrobials. The gene of interest in this study, BPSL2774, encoding BPSL2774 hypothetical protein, is a target gene that has been predicted as essential using transposon-directed insertion site sequencing technique (TraDIS). We aim to express and purify soluble GST-tagged BPSL2774 protein at sufficient concentration for future functional assays. Materials and method: The BPSL2774 gene has previously been amplified from genomic DNA of B. pseudomallei K96243 and cloned into pDEST15 (GST-tag) plasmid vector. In this work, the clone was transformed into E. coli BL21(DE3) expression strain cells for up-scaled protein preparations in 0.5 L and 1 L cultures. The auto-induction method was adopted for protein expression. GST-tag affinity chromatography was performed for protein purification and the fractions obtained were analyzed using SDS-PAGE. Results: The target protein was successfully expressed in soluble form and its highest concentration from a 0.8 mL elution fraction was at 1.38 mg/mL. Mass spectrometry analysis of 60 kDa coomassie-stained gel band cut confirmed the presence of the soluble expressed target protein, co-purified with E. coli chaperonin proteins, possibly due to their interaction with the target protein. Higher purity can be achieved through further purification steps following initial GST-tag affinity chromatography. Conclusion: The purified protein was at an acceptable purity and at sufficient concentration for use as samples in a glycosyltransferase bioluminescence assay in the near future.
format	Conference or Workshop Item
author	Mohamed Rehan, Aisyah Ujang, Hanisah Drahaman, Siti Marhamah Mat Akhir, Nor Azurah Muhamad Bunnori, Noraslinda Raih, Mohd Firdaus
author_facet	Mohamed Rehan, Aisyah Ujang, Hanisah Drahaman, Siti Marhamah Mat Akhir, Nor Azurah Muhamad Bunnori, Noraslinda Raih, Mohd Firdaus
author_sort	Mohamed Rehan, Aisyah
title	Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243
title_short	Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243
title_full	Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243
title_fullStr	Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243
title_full_unstemmed	Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243
title_sort	expression and purification of soluble recombinant gst-tagged bpsl2774 protein from burkholderia pseudomallei k96243
publishDate	2017
url	http://irep.iium.edu.my/57962/ http://irep.iium.edu.my/57962/19/57962.pdf
first_indexed	2023-09-18T21:21:56Z
last_indexed	2023-09-18T21:21:56Z
_version_	1777411939579723776

Expression and purification of soluble recombinant GST-tagged BPSL2774 protein from Burkholderia pseudomallei K96243

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