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Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology

Title
Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology
Authors
Xu, Mei LingKim, Seung CheolKim, Hyoung JinJu, WoongKim, Yun HwanKim, Hong-Jin
Ewha Authors
김승철주웅김윤환
SCOPUS Author ID
김승철scopus; 주웅scopus; 김윤환scopus
Issue Date
2017
Journal Title
PROTEIN EXPRESSION AND PURIFICATION
ISSN
1046-5928JCR Link1096-0279JCR Link
Citation
vol. 132, pp. 19 - 26
Keywords
Human papillomavirusE6 proteinSerologyCervical cancerGlutathione S-transferase
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Indexed
SCI; SCIE; SCOPUS WOS scopus
Abstract
Human papillomavirus (HPV) E6 protein is an oncoprotein with a pivotal role in cervical carcinogenesis. Expression and purification of HPV E6 from Escherichia coli (E. coli) has been difficult because of its strong hydrophobicity even when expressed as a fusion protein with glutathione S-transferase (GST). There has been no protocol suggested for purifying GST-tagged HPV E6 protein with high purity so far. Herein, we provide efficient protocol for purifying GST-HPV16 E6 protein for the first time. In the current study, the GST-tagged protein was expressed in E. coli and a purification method was designed using cation exchange chromatography followed by GST-affinity chromatography. Using physiological pH buffer during cell lysis and first cation-exchange chromatography significantly reduced yield of full-length GST-HPV16 E6 protein. It was found that using an alkaline buffer during cation-exchange chromatography was needed to obtain full length GST-HPV16 E6 protein. GST-HPV16 E6 protein recovered from the purification using alkaline condition retained its inherent p53-binding ability. Moreover, we were able to detect anti-HPV16 E6 antibodies with high sensitivity in sera from patients with cervical cancer using the GST HPV16 E6 protein. It was found that the GST-HPV16 E6 protein could be used as a coating agent to enhance the sensitivity of detection of serum anti-HPV16 E6 antibodies when treated with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). These results indicate that the two-step chromatographic purification allows obtaining high purity of GST-HPV16 E6 protein and the GST-HPV16 E6 is suitable to be used as an antigen of serology assay. (C) 2017 Elsevier Inc. All rights reserved.
DOI
10.1016/j.pep.2017.01.004
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의학전문대학원 > 의학과 > Journal papers
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