Bioprocess applications of a Sindbis virus-based temperature-inducible expression system.
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.
Boorsma
M
M
Cytos Biotechnology AG, Wagistrasse 21, CH-8952, Zurich-Schlieren, Switzerland.
Hoenke
S
S
Marrero
A
A
Fischer
R
R
Bailey
J E
JE
Renner
W A
WA
Bachmann
M F
MF
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*
D015415:Biomarkers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016899:Interferon-beta