Production of recombinant human colony stimulating factors in yeast.
Efficient yeast expression and purification systems for production of recombinant human GM-CSF, IL-3 and G-CSF have been established. Though yeast-derived production of recombinant CSFs (through the use of secretion based system) allows for generation of native molecules which can then be readily separated from fermentation broth, in many instances, natural cDNAs have had to be altered to allow for efficient expression, as well as production of a less heterogeneous product. In the case of CSFs described herein, beneficial mutations (made through site-directed mutagenesis) have included elimination of potential N-linked glycosylation sites, removal of KexII protease recognition sites (notably alterations in dibasic sequences) and elimination of extraneous cysteine residues which might complicate isolation of a homogeneous product due to intermolecular disulfide bonding.
Gillis
S
S
Department of Molecular Biology, Immunex Corporation, Seattle, Washington 98101.
Urdal
D L
DL
Clevenger
W
W
Klinke
R
R
Sassenfeld
H
H
Price
V
V
Cosman
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003115:Colony-Stimulating Factors / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D003115:Colony-Stimulating Factors; D007377:Interleukin-3; D011994:Recombinant Proteins