Expression of ovine interleukin-2 cDNA in Escherichia coli.
The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli. The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein. However, only a small proportion of this fusion protein was present in the soluble fraction. The insoluble fraction was extracted with a detergent, sarkosyl, and even though a large amount of fusion product was obtained, it would not bind to glutathione beads efficiently. Thus, only low yields of biologically active rOvIL-2 were obtained. The yields were not significantly improved when other detergents were used for extraction except for a non-ionic detergent, Zwittergent 3-14, where there was a two- to three-fold increase compared with extraction with sarkosyl. An alternative vector, pT7-7 was used with a 6 x histidine tag followed by a thrombin cleavage site at the amino terminus of the mature ovine IL-2 protein to allow affinity purification by Ni-NTA resin. A large proportion of the rOvIL-2 was partitioned to the insoluble fraction. This expression system was more useful than the pGEX-2T as large quantities of biologically active rOvIL-2 of at least 10 mg l-1 were obtained. The presence of the six histidine residues at the amino end of rOvIL-2 did not reduce its biological activity. Both systems yielded rOvIL-2 with a high specific activity of about 1 x 10(7) U mg-1 as measured by the ability to maintain proliferation of ovine ConA lymphoblasts. Recombinant OvIL-2 was active on bovine but not porcine ConA lymphoblasts.
Seow
H F
HF
Cooperative Research Centre for Vaccine Technology, Animal Health Research Laboratory, Parkville, Vic., Australia.
Mucha
M J
MJ
Hurst
L
L
Rothel
J S
JS
Wood
P R
PR
D000818:Animals; D002417:Cattle; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D012756:Sheep; D013045:Species Specificity; D013552:Swine
D018076:DNA, Complementary; D007376:Interleukin-2; D011994:Recombinant Proteins