THE CLONING, EXPRESSION AND PURIFICATION OF CERVINE INTERLEUKIN-2

The cloning, sequencing and expression of cervine interleukin 2 (IL-2) is described, Cervine IL-2 cDNA is 489 base pairs long and shows high homology to bovine and ovine IL-2 (similar to 94%) with lower homolog ies to human (50%) and mouse (53%). The predicted protein sequence is 162 amino acids long with a signal sequence containing 20 amino acids, A molecular weight of 16 273 Da was predicted for the mature protein, The expression plasmid pTRXFUS was redesigned to allow recombinant pr oteins to be expressed at high levels in a soluble form and subsequent ly affinity purified, This new plasmid, pTRXHIS, has been used to expr ess the first cervine cytokine, IL-2, The fusion of the cervine IL-2 g ene to the thioredoxin gene (TRX) stabilizes the recombinant product a llowing the high expression of soluble IL-2, A polyHis (6 x Histidines ) tag has been inserted between the two fusion partners which allows t he fusion product to be affinity purified on a nickel-nitrilo-tri-acet ic acid (Ni-NTA) column, The purified cervine IL-2 fusion protein was shown to be biologically active despite the presence of the TRX at the amino terminus, The TRX can be removed enzymatically with enterokinas e releasing the biologically active IL-2 molecule, This expression sys tem has several features that are useful in producing and purifying la rge quantities of biologically active cytokines. (C) 1996 Academic Pre ss Limited