Journal: Protein Expr Purif / Year: 2017 Title: Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli. Authors: Daning Wang / Fei Fan / Zhihai Li / Xinlin Liu / Shuo Song / Shuangping Wei / Maozhou He / Yahua Lin / Zhongyi Li / Minxi Wei / Hai Yu / Ying Gu / Shaowei Li / Ningshao Xia / Abstract: Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be ...Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be prevented by vaccination with HPV vaccines derived from eukaryotic expression systems. Here, we report the soluble expression of the major capsid protein L1 of HPV31, a dominant carcinogenic HPV genotype, in Escherichia coli. HPV31 L1 protein and its elongated form (L1+) were observed in SDS-PAGE and CE-SDS analysis, generated by the native HPV31 L1 gene with a TAA stop codon. Replacing the TAA with TAG but not TGA could completely terminate protein translation. Mass spectrometry sequencing showed that L1+ comprised L1 with a C-terminal extension of 38 amino acids (aa). RNA folding analysis revealed that the unfaithful L1+ expression may result from translational read-through, as TAG is more stable and accessible than the other stop codons. The 38-aa elongated fragment perturbs self-assembly of HPV31 L1+, as shown in size and morphology analyses. By 3D cryo-electron microscopy structure determination, we show self-assembly of purified HPV31 L1 (TAG) VLPs into T = 7 icosahedral symmetry particles, resembling the native HPV virion. Finally, through additional characterization and antigenicity/immunogenicity assays, we verified that the E.coli-derived HPV31 VLPs are an ideal immunogen for HPV vaccine development. Our findings outline a codon optimization stratagem for protein expression and provide a method for the in-depth investigation of prokaryotic translation regulation.
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Feb 10, 2018
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