Premium
Factors influencing recombinant protein yields in an insect cell–bacuiovirus expression system: Multiplicity of infection and intracellular protein degradation
Author(s) -
Licari P.,
Bailey J. E.
Publication year - 1991
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260370306
Subject(s) - polyhedrin , multiplicity of infection , sf9 , titer , recombinant dna , biology , autographa californica , baculoviridae , intracellular , heterologous , microbiology and biotechnology , protein biosynthesis , recombinant virus , virology , virus , spodoptera , gene , biochemistry
The insect cell (Sf9)‐baculovirus (AcNPV) expression system was employed for the synthesis of β‐galactosidase, a model heterologous protein. In the recombinant virus studied, the lacZ gene is fused to a portion of the polyhedrin structural gene and is under the control of the polyhedrin promoter. The effect of the multiplicity of infection (MOI) on product titer was determined by infecting cells with MOI values ranging from 0 to 100 and monitoring the production of β‐galactosidase with time. The relationship between final product titer and MOI was dependent on the growth phase of the cells prior to infection. The final product titer from cells infected in the early exponential phase was relatively independent of MOI. For cells infected in late‐exponential phase there was a logarithmic relationship between the final β‐galactosidase titer and the MOI used, with the highest MOI studied resulting in greatest protein synthesis. The synthesis and degradation rates of β‐galactosidase were investigated by a pulse‐chase technique using L ‐[ 35 S]‐methionine. At 24 h postinfection, the degradation rate is of the same order of magnitude as the synthesis rate. However, the synthesis rate of β‐galactosidase increases dramatically at 96 h postinfection. During this later period, the degradation rate is negligible. Although degradation of recombinant protein occurs in this system, degradation activity declines as infection proceeds and is insignificant late in intention when recombinant protein expression is intense.