Open AccessProduction and purification of recombinant human interleukin‐5 from yeast and baculovirus expression systems
Author(s) -
INGLEY Evan,
CUTLER Robert L.,
FUNG MingChiu,
SANDERSON Colin J.,
YOUNG Ian G.
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb15858.x
Subject(s) - sf9 , recombinant dna , microbiology and biotechnology , complementary dna , biology , fusion protein , yeast , gene , biochemistry , spodoptera
A c DNA for human interleukin‐5 (hIL‐5) was created from the hIL‐5 gene using site‐directed mutagenesis to splice out the introns in vitro . This c DNA was expressed in yeast and baculovirus systems, utilizing in both cases an in‐frame fusion to the pre sequence of the α‐mating‐type factor to direct secretion. The highest level of production was achieved from Sf9 cells using a baculovirus vector in serum‐containing medium (2.7 mg/l), whereas in serum‐free medium ten times less hIL‐5 was produced. In the yeast system much lower levels of hIL‐5 were produced (12.5 μg/l). Recombinant hIL‐5 was purified to homogeneity from serum‐free baculovirus cultures. The rhIL‐5 consisted of a 30‐kDa homodimer linked by disulfide bridging. The purified recombinant protein had a specific activity on murine BCL 1 cells of 1.5x10 4 U/mg, of 3x10 5 U/mg in the murine eosinophil differentiation factor assay, and 2.4x10 7 U/mg in a human peripheral eosinophil maintenance assay.