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Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology
- Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology
- Xu, Mei Ling; Kim, Seung Cheol; Kim, Hyoung Jin; Ju, Woong; Kim, Yun Hwan; Kim, Hong-Jin
- Ewha Authors
- 김승철; 주웅; 김윤환
- SCOPUS Author ID
- 김승철; 주웅; 김윤환
- Issue Date
- Journal Title
- PROTEIN EXPRESSION AND PURIFICATION
- 1046-5928; 1096-0279
- vol. 132, pp. 19 - 26
- Human papillomavirus; E6 protein; Serology; Cervical cancer; Glutathione S-transferase
- ACADEMIC PRESS INC ELSEVIER SCIENCE
- SCI; SCIE; SCOPUS
- 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.
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