Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16.
The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.
Yang
Dan-Hui
DH
Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ont., Canada N1G 2W1.
Wildeman
Alan G
AG
Sharom
Frances J
FJ
D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D027383:Papillomaviridae / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000086082:Viroporin Proteins
D008081:Liposomes; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins