Codon optimization of the HPV-16 E5 gene enhances protein expression.
The human papillomavirus type 16 (HPV-16) E5 protein is an 83-amino-acid, hydrophobic polypeptide that has been localized to intracellular membranes when overexpressed in COS-1 cells. While the HPV-16 E5 protein appears to modulate endosomal pH and signal transduction pathways, genetic analysis of its biological activities has been hampered by low (usually nondetectable) levels of expression in stable cell lines. Sequence analysis of the native HPV-16 E5 gene revealed that infrequent-use codons are used for 33 of its 83 amino acids and, in an effort to optimize E5 expression, we converted these codons to those more common in mammalian genes. The modified gene, 16E5*, generated protein levels that were six- to ninefold higher than those of wild-type HPV-16 E5, whereas the levels of mRNA were unchanged. 16E5* protein was detectable in keratinocytes by immunoblotting, immunoprecipitation, and immunofluorescence techniques and formed disulfide-dependent dimers and higher-order oligomers. Unlike the bovine papillomavirus E5 protein, which is present in the Golgi, 16E5* was localized primarily to the endoplasmic reticulum and its expression reduced the in vitro life span of keratinocytes.
Disbrow
Gary L
GL
Department of Pathology, Georgetown University Medical School, Georgetown University Medical Center, Preclinical Science Building, Room GR10C, Washington, DC 20057, USA.
Sunitha
Iruvanti
I
Baker
Carl C
CC
Hanover
John
J
Schlegel
Richard
R
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D003062:Codon; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000821:virology; D051379:Mice; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D011995:Recombination, Genetic; D000086082:Viroporin Proteins
D003062:Codon; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins