Codon optimization of the HPV-16 E5 gene enhances protein expression

Area of Science:

• Molecular Biology
• Virology
• Cell Biology

Background:

• The human papillomavirus type 16 (HPV-16) E5 protein is crucial for viral oncogenesis but is difficult to study due to low expression levels.
• Previous studies indicate HPV-16 E5 modulates endosomal pH and signal transduction pathways.
• Genetic analysis of HPV-16 E5 biological activities has been limited by low expression in stable cell lines.

Purpose of the Study:

• To overcome low expression issues of the HPV-16 E5 protein.
• To optimize the expression of HPV-16 E5 protein for further biological studies.
• To investigate the localization and effects of enhanced HPV-16 E5 expression in keratinocytes.

Main Methods:

• Sequence analysis of the native HPV-16 E5 gene to identify infrequent-use codons.
• Codon optimization of the HPV-16 E5 gene (16E5*) by replacing rare codons with more common mammalian codons.
• Detection and analysis of 16E5* protein levels and localization in keratinocytes using immunoblotting, immunoprecipitation, and immunofluorescence.

Main Results:

• Codon optimization of the HPV-16 E5 gene resulted in a six- to ninefold increase in protein levels without altering mRNA levels.
• The modified 16E5* protein was detectable in keratinocytes and formed disulfide-dependent dimers and oligomers.
• 16E5* localized primarily to the endoplasmic reticulum and reduced the in vitro lifespan of keratinocytes.

Conclusions:

• Codon optimization is an effective strategy to enhance the expression of the HPV-16 E5 protein.
• Enhanced HPV-16 E5 expression allows for detailed molecular and cellular analysis.
• The endoplasmic reticulum localization of HPV-16 E5 and its impact on keratinocyte lifespan warrant further investigation.