Biochemical characterization of murine leukaemia inhibitory factor produced by Krebs ascites and by yeast cells.
A cDNA library was constructed using mRNA from Krebs ascites tumor cells that was shown by Northern blot hybridization to contain mRNA for murine leukemia inhibitory factor (LIF). This library was screened with an oligonucleotide corresponding to the 3' end of a partial LIF cDNA clone, and an overlapping cDNA clone isolated. Nucleotide sequence analysis of this latter clone allowed the complete sequence of LIF to be derived. A cDNA molecule encoding the entire mature LIF protein was installed in a yeast expression vector, and LIF produced up to about 100 ng/ml in the growth medium. The LIF produced by yeast cells has the same biologic properties as native LIF and competes with native 125I-LIF for binding to specific cellular receptors. Two forms of native LIF, distinguishable by their chromatographic behavior on DEAE-Sepharose, were converted by neuraminidase treatment to a form with similar chromatographic behavior, suggesting that the major difference between these two species is the content of sialic acid on the carbohydrate portion. Moreover, yeast-derived recombinant LIF appears to display a different pattern of glycosylation to both forms of native LIF. From in vitro experiments, we conclude that the nature of the glycosylation is not crucial to biologic activity.
Gough
N M
NM
Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, Australia.
Hilton
D J
DJ
Gearing
D P
DP
Willson
T A
TA
King
J A
JA
Nicola
N A
NA
Metcalf
D
D
D000818:Animals; D002287:Carcinoma, Krebs 2 / Q000235:genetics*; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D006031:Glycosylation; D006131:Growth Inhibitors / Q000096:biosynthesis* / Q000502:physiology; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D006131:Growth Inhibitors; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D011994:Recombinant Proteins