Functional oligomerization of purified human papillomavirus types 16 and 6b E7 proteins expressed in Escherichia coli (original) (raw)
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Virology, 1998
Major capsid protein L1 of HPV16 was produced in a fused form in Escherichia coli using an inducible expression system. The protein formed insoluble aggregations (inclusion bodies) and the yield was more than 10% of total cell proteins. The inclusion bodies were isolated and solubilised with 8 M urea and the L1 proteins were purified by chromatographic separation. Following removal of the urea by gradual dialysis, the denatured L1 proteins spontaneously renatured and subsequently assembled into polymorphologic aggregations in vitro. Electron microscopy showed that the assembled material included structures resembling native empty capsids as well as incompletely formed capsids. After separation from the pool of polymorphologic structures by sucrose gradient sedimentation, the correctly formed virus-like particles (VLE. coliPs) were recognised by a HPV16 type-specific, conformational-dependent monoclonal antibody in an ELISA. This system offers not only a model for investigation of the intrinsic interactions that occur during L1 assembly, but also a potential route for convenient manufacture of highly purified VLP vaccines.
Journal of Genetic Engineering and Biotechnology
Background A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. Results The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning...
Iranian journal of cancer prevention, 2012
Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm. In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/T-E6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression ve...
The human papillomavirus type 16 (HPV-16) E7 gene encodes a multifunctional oncoprotein that can subvert multiple cellular regulatory pathways. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional cellular targets that contribute to its transforming potential. We isolated HPV-16 E7 associated cellular protein complexes by tandem affinity purification and mass spectrometry and identified the 600-kDa retinoblastoma protein associated factor, p600, as a cellular target of E7. Association of E7 with p600 is independent of the pocket proteins and is mediated through the N terminal E7 domain, which is related to conserved region 1 of the adenovirus E1A protein and importantly contributes to cellular transformation independent of pRB binding. Depletion of p600 protein levels by RNA interference substantially decreased anchorage-independent growth in HPV-positive and -negative human cancer cells. Therefore, p600 is a cellular target of E7 that regulates cellular pathways that contribute to anchorage-independent growth and cellular transformation.
Dimerization of the Human Papillomavirus E7 Oncoproteinin Vivo
Virology, 1995
We have used a yeast two-hybrid system to show that human papillomavirus E7 proteins can form oligomeric complexes in vivo. The carboxyl-terminal cysteine-rich metal-binding domain is critical for this activity although amino-terminal sequences also contribute to oligomerization. Our experiments also reveal that E7 possesses an intrinsic transcription activation activity in yeast, which resides in the amino terminus of the protein. ᭧
Journal of Virology
The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to ...
Virology, 1992
Human papillomavirus type 16 (HPV 16) is often found in cervical carcinomas, while HPV 6 is frequently associated with benign genital lesions. We have compared the abilities of the E7 transforming proteins of HPV 6 and 16 to transform various established and primary rodent cells by using the same heterologous promoter system. HPV 16 E7 efficiently induced anchorage-independent growth of all the rodent cell lines tested and immortalized or cooperated with ras in transforming primary rat cells. On the other hand, the transforming activity of HPV 6 E7 was lower and was restricted. By construction of chimeras of HPV 6 and 16 E7, we found that the difference in transforming activity between the two E7 proteins was mainly determined by the difference in their 30 N-terminal amino acid residues, although some activities seem to be slightly affected by differences in their residual C-terminal portions. o 1992 Academic Press. Inc.
Journal of Virology, 2013
We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.
Characterization of functional HPV-16 E7 protein produced in Escherichia coli
Journal of Biological Chemistry, 1992
Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are contributing factors in the pathogenesis of human cervical cancer. The HPV E7 gene is transcriptionally active in these diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherichia coli. The isolated E7 protein contains zinc in a 1:l molar ratio. X-ray absorption fine structure studies demonstrated that the zinc is coordinated by 4 sulfur ligands. We sequentially derivatized the E7 cysteines to differentiate between solvent-exposed, metal-bound, and disulfide-associated cysteines. Our results demonstrate that CysZ4 and Cyses are accessible to solvent, while cysteines in the two conserved Cys-X-X-Cys motifs are likely involved in binding zinc. We observed no evidence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested. Nearly half of the known human papilloma viruses (HPVs)' can infect the genital mucosa and produce benign epithelial lesions. Epidemiological and molecular genetic studies have demonstrated that only a few of the HPV isolates that infect the genital mucosa are associated with most (75-100%) cases of cervical cancer (1). In these cases two viral genes, E6 and E7, are transcriptionally active (2). The most common HPV strain associated with cervical neoplasia is HPV-16. The HPV-16 E6 and E7 genes together have been shown to be necessary and sufficient for transformation of primary human keratinocytes (3). The HPV-16 E7 gene is able to transform established rodent fibroblasts (4), and, in conjunction with ras, primary rat epithelial cells (5). The HPV-16 E7 gene encodes a protein of 98 amino acids (6) which shares a region of homology (amino acids 2-35) with the adenovirus E1A protein and simian virus-40 large T antigen. E7, like ElA, can transactivate transcription from ~~ ~ * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.