title: Bioprocess applications of a Sindbis virus-based temperature-inducible expression system.
abstract: The production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature-regulatable Sindbis virus replicon-based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth-inhibiting, anti-viral protein interferon beta (beta-IFN). Selected clones were analyzed for temperature-dependent beta-IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 x 10(5) IU/10(6) cells/day (0.5 microg/10(6) cells/day) in suspension cultures and over 1.5 x 10(6) IU/mL/day (7.5 microg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine-tagged beta-IFN purified from T-flask cultures was highly glycosylated and showed high specific activity. beta-IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS-inducible expression system for the production of high-quality glycoproteins in different reactors.
authors.affiliation: Cytos Biotechnology AG, Wagistrasse 21, CH-8952, Zurich-Schlieren, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Boorsma
authors.forename: S
authors.initials: S
authors.lastname: Hoenke
authors.forename: A
authors.initials: A
authors.lastname: Marrero
authors.forename: R
authors.initials: R
authors.lastname: Fischer
authors.forename: J E
authors.initials: JE
authors.lastname: Bailey
authors.forename: W A
authors.initials: WA
authors.lastname: Renner
authors.forename: M F
authors.initials: MF
authors.lastname: Bachmann
mesh_terms: D000818:Animals; D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics*; D013696:Temperature; D014162:Transfection / Q000379:methods*
chemical_list: D015415:Biomarkers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016899:Interferon-beta