A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.
Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells.
Casales
Erkuden
E
Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
Aranda
Alejandro
A
Quetglas
Jose I
JI
Ruiz-Guillen
Marta
M
Rodriguez-Madoz
Juan R
JR
Prieto
Jesus
J
Smerdou
Cristian
C
D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism / Q000627:therapeutic use; D012672:Semliki forest virus / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D016207:Cytokines; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; C092073:cardiotrophin 1