title: Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli.
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 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.
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Daning
authors.initials: D
authors.lastname: Wang
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Fan
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Zhihai
authors.initials: Z
authors.lastname: Li
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Xinlin
authors.initials: X
authors.lastname: Liu
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Song
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Shuangping
authors.initials: S
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Maozhou
authors.initials: M
authors.lastname: He
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Yahua
authors.initials: Y
authors.lastname: Lin
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Li
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Minxi
authors.initials: M
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Gu
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China. Electronic address: shaowei@xmu.edu.cn.
authors.forename: Shaowei
authors.initials: S
authors.lastname: Li
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, 361102, China; National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, 361102, China.
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D036022:Capsid Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D058952:Human papillomavirus 31 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D053918:Papillomavirus Vaccines / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D018388:Codon, Terminator; C114580:HPV L1 protein, Human papillomavirus; D009856:Oncogene Proteins, Viral; D053918:Papillomavirus Vaccines; D011994:Recombinant Proteins
keywords: Escherichia coli; Expression; Human papillomavirus; Major capsid protein; TAG stop codon