Open AccessProduction and purification of recombinant human interleukin‐6 secreted by the yeast Saccharomyces cerevisiae
Author(s) -
GUISEZ Yves,
TISON Bernard,
VANDEKERCKHOVE Joël,
DEMOLDER Jan,
BAUW Guy,
HAEGEMAN Guy,
FIERS Walter,
CONTRERAS Roland
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.tb16004.x
Subject(s) - saccharomyces cerevisiae , secretion , signal peptide , protease , yeast , secretory protein , recombinant dna , mutant , biology , biochemistry , microbiology and biotechnology , gene , chemistry , enzyme
The coding region of the human interleukin‐6 (hIL6) gene was fused to the prepro secretion signal of the α‐mating factor gene in several yeast host strains. It was found that the KEX‐2 protease was unable to cleave the prepro‐Lys‐Arg‐Pro‐IL6 sequence, but that unspecific cleavage of the precursor protein had occurred. The prepro‐Lys‐Arg‐Ala‐Pro‐IL6 sequence, however, was correctly recognized and cleaved by the KEX‐2 protease, and IL6 was efficiently secreted into the culture medium. The N‐terminal Ala‐Pro peptide was removed during processing by wild‐type yeast strains, but was retained in a ste13 mutant. IL6 as well as the aberrant proteins were not glycosylated. The transformed cells could secrete up to 30 μg/ml IL6. The protein was purified from the medium to homogeneity by ion‐exchange chromatography and gel filtration, and had a specific activity of about 2 × 10 8 IU/mg in a proliferation assay.