279b3231c8ad3d20292024c5fb0f1941 pmc_34834382.pdf 9bffeebf0068a612f9b6bb00c0f6b36380fb51d3 pmc_34834382.pdf f91a3a8a45561b221be54eada4fa5754fe07ee7e24974781f72f2363d929705f pmc_34834382.pdf Title: Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles Subject: Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris. Keywords: HPV; HIV; virus-like particle; chimeric; vaccine; yeast; Pichia pastoris; purification Author: Yoshiki Eto, Narcís Saubi, Pau Ferrer and Joan Joseph-Munné Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Nov 24 10:40:16 2021 CET ModDate: Wed Nov 24 11:06:03 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 15614672 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LWJOET+VnURWPalladioL Type 1 Custom yes yes no 10 0 LQBRGK+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 PLJLSL+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 CDKKOC+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 ZGAQSB+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 YSPQJI+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 41 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 69 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 73 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 77 0 SPXLNH+PazoMath Type 1 Builtin yes yes no 92 0 QGYEQT+CMSY10 Type 1 Builtin yes yes no 96 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 100 0 BBIOBF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 147 0 BBIOBG+PalatinoLinotype CID TrueType Identity-H yes yes yes 150 0 BBIOBH+PalatinoLinotype TrueType WinAnsi yes yes no 156 0 BBIOBJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 159 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 172 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 183 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 187 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 191 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 205 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 209 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 213 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 217 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 02:54:18 CEST RepresentationInformation: pmc_34834382.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:06:22 CEST Size: 15614672 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 445 FreeObjects: 9 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Bacterial and Yeast Strains, and Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Design and Construction of Recombinant P. pastoris X-33 Strain Expressing Chimeric L1P18 Protein Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Production and Purification of L1P18 VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Immunodot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Sodium Dodecyl Sulphate Polyacrylamide gel Electrophoresis and Western Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Transmission Electron Microscopy of L1P18 VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Heterologous Protein and Total Protein Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Ethics Statement Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Construction of Recombinant P. pastoris X-33-L1P18I10 Strain Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Genetic and Phenotypic Characterization of Recombinant X-33-L1P18I10 Yeast Strain Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Production and Purification of Chimeric L1P18 VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Characterization of Purified L1P18 VLPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Info: Title: Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles Author: Yoshiki Eto, Narcís Saubi, Pau Ferrer and Joan Joseph-Munné Subject: Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris. 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