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 (approximately 94%) with lower homologies 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 16273 Da was predicted for the mature protein. The expression plasmid pTRXFUS was redesigned to allow recombinant proteins to be expressed at high levels in a soluble form and subsequently affinity purified. This new plasmid, pTRXHIS, has been used to express the first cervine cytokine, IL-2. The fusion of the cervine IL-2 gene to the thioredoxin gene (TRX) stabilizes the recombinant product allowing the high expression of soluble IL-2. A polyHis (6 x Histidines) tag has been inserted between the two fusion partners which allows the fusion product to be affinity purified on a nickel-nitrilo-tri-acetic 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 enterokinase releasing the biologically active IL-2 molecule. This expression system has several features that are useful in producing and purifying large quantities of biologically active cytokines.
Lockhart
E
E
Department of Microbiology, University of Otago, Dunedin, New Zealand.
Griffin
J F
JF
Chinn
N
N
Lavallie
E
E
Buchan
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003670:Deer; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017421:Sequence Analysis
D007376:Interleukin-2; D011994:Recombinant Proteins