The E6 variant proteins E6I-E6IV of human papillomavirus 16: expression in cell free systems and bacteria and study of their interaction with p53.
Several species of alternatively spliced mRNAs are transcribed from the E6 gene region of human papillomavirus (HPV) 16. These have the coding capacity for either the full length E6 of 151 amino acids (aa) or four truncated variants, E6I-E6IV, of 43-64 aa. As the first step to identify the putative E6 variants and their functions, we generated cDNAs corresponding to the various E6 open reading frames (ORF) and examined their expression employing in vitro transcription/translation systems and the bacterial pET system. In wheat germ extract, in vitro translation resulted in the production of all five proteins, E6 and E6I-E6IV. These proteins were also expressed as stable fusion proteins from the pET16b and pET17 x b vectors in Escherichia coli. Mobilites of the E6 variant proteins on SDS-acrylamide gels were consistent with their predicted sizes. The authenticity of the synthesized proteins was confirmed by immunoprecipitation with specific antibodies directed against epitopes in the N-terminal portion of E6 as well as antibodies raised against the individual variant proteins produced in E. coli. In rabbit reticulocyte lysate, however, only the full length E6 and the E6IV variant were synthesized. This could be due to inefficient translation as well as lower stability of the short variants. E6I-III, in reticulocyte lysate (RTL). The ability of the E6 variants to associate with p53 and target its proteolytic degradation in vitro, was examined in coimmunoprecipitation assays, using in vitro synthesized proteins and monoclonal antibodies to p53. Results of these assays indicated that only the full length E6 efficiently binds to and promotes the degradation of p53. The E6 variants E6I-E6IV, although able to associate with p53 at a low efficiency, were unable to target its degradation.
Shally
M
M
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Alloul
N
N
Jackman
A
A
Muller
M
M
Gissmann
L
L
Sherman
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53