Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor.
Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3 x 10(6)U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.
Yamamoto
Akira
A
Nippon Institute for Biological Science, Shin-Machi 9-2221-1, Ome, Tokyo 198-0024, Japan. yamagen@nibs.or.jp
Iwata
Akira
A
Saitoh
Toshiki
T
Tuchiya
Kotaro
K
Kanai
Tomoko
T
Tsujimoto
Hajime
H
Hasegawa
Atsuhiko
A
Ishihama
Akira
A
Ueda
Susumu
S
D000818:Animals; D002415:Cats; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D006098:Granulocytes / Q000187:drug effects; D007958:Leukocyte Count / Q000662:veterinary; D008264:Macrophages / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology; D011994:Recombinant Proteins; D013997:Time Factors
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor