title: Two-step chromatographic purification of glutathione S-transferase-tagged human papillomavirus type 16 E6 protein and its application for serology.
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 (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.
authors.affiliation: Laboratory of Virology, College of Pharmacy, Chung-Ang University, Seoul, South Korea.
authors.forename: Mei Ling
authors.initials: ML
authors.lastname: Xu
authors.affiliation: Department of Obstetrics and Gynecology, Ewha Womans University College of Medicine, Seoul, South Korea.
authors.forename: Seung Cheol
authors.initials: SC
authors.lastname: Kim
authors.affiliation: Laboratory of Virology, College of Pharmacy, Chung-Ang University, Seoul, South Korea.
authors.forename: Hyoung Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Obstetrics and Gynecology, Ewha Womans University College of Medicine, Seoul, South Korea.
authors.forename: Woong
authors.initials: W
authors.lastname: Ju
authors.affiliation: Department of Obstetrics and Gynecology, Ewha Womans University College of Medicine, Seoul, South Korea.
authors.forename: Yun Hwan
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Laboratory of Virology, College of Pharmacy, Chung-Ang University, Seoul, South Korea. Electronic address: hongjink@cau.ac.kr.
authors.forename: Hong-Jin
authors.initials: HJ
authors.lastname: Kim
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D005982:Glutathione Transferase
keywords: Cervical cancer; E6 protein; Glutathione S-transferase; Human papillomavirus; Serology