E. coli expression and purification of human and cynomolgus IL-15.
The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)(6)-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8M urea. Use of a multi-component screen identified the optimal conditions for folding (His)(6)-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3(-) CD8+ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.
AstraZeneca R&D Charnwood, Bioscience Department, Bakewell Road, Loughborough LE115RH, UK.
Alison
A
Ward
Malcolm
M
Anderson
Robert I
RI
Craggs
Justine
J
Maltby
Caroline
C
Grahames
Rick A
RA
Davies
Donna
D
Finch
Debbie
D
Pattison
Heather
H
Oakes
Philip R
PR
Mallinder
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D002850:Chromatography, Gel; D003956:Dialysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019409:Interleukin-15 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D037181:Lectins, C-Type; D008252:Macaca fascicularis / Q000235:genetics; D008970:Molecular Weight; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015854:Up-Regulation; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D019409:Interleukin-15; D037181:Lectins, C-Type; D011994:Recombinant Proteins; D000099192:CD69 Antigens