Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology
- 주제(키워드) Cervical cancer , E6 protein , Glutathione S-transferase , Human papillomavirus , Serology
- 등재 SCIE, SCOPUS
- 발행기관 Academic Press Inc.
- 발행년도 2017
- 총서유형 Journal
- URI http://www.dcollection.net/handler/ewha/000000141722
- 본문언어 영어
- Published As http://dx.doi.org/10.1016/j.pep.2017.01.004
- 저작권 이화여자대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
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 (β-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. © 2017 Elsevier Inc.
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