RESUMO
Occasionally, only a small percentage of recombinant proteins produced in the baculovirus expression system are functionally active. We had previously shown that the majority of protein kinase C-delta (PKC-delta) produced in insect cells was inactive; less than 1% of the expressed enzyme had lipid-dependent kinase activity. In this report, we have attempted to optimize the production of a catalytically active PKC-delta. Under optimum conditions, we were able to increase the levels of PKC-delta from 10-20% to about 65% of the total cellular protein; however, there was no increase in the levels of catalytically active enzyme. Expression of PKC-delta as a fusion protein or as a secreted protein also met with limited success. Under all conditions, expression of PKC-delta proteins under control of the strong polyhedrin promoter resulted in the production of large amounts of inactive enzyme. Expression under the control of the basic protein promoter, Pcor, resulted in the reduction of the levels of recombinant protein by a factor of about four, but the PKC-delta enzyme produced under these conditions was 10- to 15-fold more active. Thus, the earlier temporal expression of PKC-delta in insect cells resulted in the production of more active enzyme.
Assuntos
Isoenzimas/biossíntese , Proteína Quinase C/biossíntese , Animais , Sequência de Bases , Células Cultivadas , Vetores Genéticos , Isoenzimas/genética , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Proteínas de Matriz de Corpos de Inclusão , Ésteres de Forbol/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteína Quinase C/genética , Proteína Quinase C-delta , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Spodoptera/citologia , Proteínas Virais/genética , Proteínas Estruturais ViraisRESUMO
A simple and relatively inexpensive stirred-vessel system for the large-scale growth of insect cells (Sf9) is described. Sf9 cell growth in a stirred-vessel fermentor and an airlift fermentor were compared on the basis of maximum cell density and average population doubling time. Also, both fermentor systems were compared with respect to the large-scale production of a recombinant human protein (protein kinase C-eta). No significant differences in Sf9 cell growth or protein expression levels were apparent between the two fermentor systems. However, large differences in cost and scale-up of each system are discussed with respect to the large-scale production of recombinant proteins.