Increased production of active human beta(2)-adrenergic/G(alphas) fusion receptor in Sf-9 cells using nutrient limiting conditions.
Using the baculovirus/insect-cell expression vector system, we succeeded in obtaining a high yield of active human beta(2)-adrenergic receptor/G(alphas) fusion protein. This was achieved following high cell density production under nutrient-limiting conditions using a very low multiplicity of infection (MOI). This approach was found to significantly reduce inactive protein accumulation that occurred when production was done using conventional high MOI procedures. The maximum specific and volumetric yields of active receptor using this strategy increased by factors of two- and sixfold, respectively. Our results suggest that the increase in the ratio of active/total protein produced results from production under nutrient limitation. Since low multiplicity of infection offers many advantages for large-scale applications, we suggest that this simple production method should be considered when optimizing expression of G-protein-coupled receptors and other complex proteins.
Carpentier
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E
Bioprocess sector, Biotechnology Research Institute, 6100 Royalmount avenue, Montreal, Quebec, H4P 2R2, Canada
Lebesgue
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D
Kamen
A A
AA
Hogue
M
M
Bouvier
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M
Durocher
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003470:Culture Media; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007118:Immunoassay; D007313:Insecta; D011485:Protein Binding; D021122:Protein Subunits; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014161:Transduction, Genetic
D003470:Culture Media; D021122:Protein Subunits; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D020962:Heterotrimeric GTP-Binding Proteins