Expression and purification of recombinant ovine interleukin-1 beta from Escherichia coli.
An expression vector bearing the gene segment encoding the mature form of ovine interleukin-1 beta (OvIL-1 beta) was constructed. This vector provided a rapid method for obtaining Escherichia coli derived recombinant OvIL-1 beta (rOvIL-1 beta) using the expression plasmid pGEX-2T. The level of expression of fusion protein in the soluble fraction was approximately 20% of the total accumulated proteins. Affinity purification by glutathione-Sepharose yielded a fusion protein and subsequent thrombin cleavage of this material yielded rOvIL-1 beta. The specific activity of the purified recombinant protein was 10(3)-10(4) times higher than the fusion protein. The rOvIL-1 beta was 10-100 times more potent than human interleukin-1 beta (HuIL-1 beta) in an ovine thymocyte proliferation assay, although they were of equal potency in the NOB-1/CTLL assay. This simple purification method, which produces purified rOvIL-1 beta with a high specific activity (approximately 10(8) U mg-1), will now make it possible to evaluate the in vivo effects of IL-1 beta in sheep.
Seow
H F
HF
CSIRO Division of Animal Health, Animal Health Research Laboratory, Parkville, Vic., Australia.
Rothel
J S
JS
Wood
P R
PR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012756:Sheep / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology
D017931:DNA Primers; D007375:Interleukin-1; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins