title: Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.
abstract: Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.
authors.affiliation: Nature Technology Corporation, Lincoln, NE 68521, USA.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Luke
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Carnes
authors.forename: Ping
authors.initials: P
authors.lastname: Sun
authors.forename: Clague P
authors.initials: CP
authors.lastname: Hodgson
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
authors.forename: James A
authors.initials: JA
authors.lastname: Williams
mesh_terms: D000345:Affinity Labels; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D005285:Fermentation; D005408:Flagellin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006389:Hemagglutinins, Viral / Q000235:genetics; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology; D007252:Influenza Vaccines / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013696:Temperature
chemical_list: D000345:Affinity Labels; D000914:Antibodies, Viral; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006389:Hemagglutinins, Viral; D007252:Influenza Vaccines; D011993:Recombinant Fusion Proteins; D005408:Flagellin