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Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16

Yang, Dan-Hui, Wildeman, Alan G, Sharom, Frances J·2003·Protein Expression and Purification
DOI 10.1016/s1046-5928(03)00049-4
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Abstract

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 a…

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Metadata

Authors
Yang, D.-H., Wildeman, A. G., & Sharom, F. J.
Venue
Protein Expression and Purification
Publisher
Elsevier BV
Published
2003-07
Volume
30
Issue
1
Pages
1-10
ISSN
1046-5928
DOI
10.1016/s1046-5928(03)00049-4
Cited by
15
References
0
Language
en
Licence
www.elsevier.com/tdm/userlicense/1.0/ (tdm)

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CoChat. (2026). Citation check: Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16. https://app.cochat.ai/papers/10.1016/s1046-5928(03)00049-4
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Yang, D.-H., Wildeman, A. G., & Sharom, F. J. (2003). Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16. Protein Expression and Purification. https://doi.org/10.1016/s1046-5928(03)00049-4