Interleukin-1 receptor antagonist: characterisation of its gene expression in rabbit tissues and large-scale expression in eucaryotic cells using a baculovirus expression system.
The gene expression of rabbit interleukin-1 receptor antagonist (RbIL-lra) was examined in rabbit tissues. RNA was isolated from heart, lung, kidney, muscle, liver, spleen, brain, and peripheral blood monocytes (PBMs), and RbIL-lra mRNA was identified as a single species by Northern analysis using a RbIL-lra probe. RbIL-lra was abundantly expressed in lung, brain, heart, and liver, expressed at low levels in spleen, and undetectable in kidney and unstimulated PBMs. Expression of large scale recombinant production of RbIL-lra was achieved by subcloning the cDNA into a baculovirus expression vector. Recombination of this vector was completed with the BacPAK6 baculovirus genome. The recombinant virus, containing the RbIL-lra cDNA, was used to infect Spodoptera frugiperda (Sf21) insect cells in a spinner flask system and in monolayers in cell culture flasks. Recombinant rabbit IL-lra (rRbIL-lra) was secreted into the culture medium in this system at very high levels (35 mg/l). The protein was identified by reducing SDS-PAGE electrophoresis, was variably glycosylated and had a molecular weight between 19-25 kDa. It was then purified by size exclusion HPLC on a Du Pont Gf-250 column. The rRbIL-lra was demonstrated to be functionally active by inhibiting recombinant human IL-1 alpha in a mouse thymocyte proliferation assay. 20 ng/ml (6.7 U/ml) of rRbIL-lra inhibited 95% of the activity of 2 ng/ml IL-1 alpha.
Apostolopoulos
J
J
Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia.
Ross
S
S
Davenport
P
P
Matsukawa
A
A
Yoshinaga
M
M
Tipping
P G
PG
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression / Q000276:immunology*; D005822:Genetic Vectors / Q000378:metabolism; D053590:Interleukin 1 Receptor Antagonist Protein; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D011817:Rabbits; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D014781:Viscera / Q000378:metabolism
C507763:IL1RN protein, human; C507764:Il1rn protein, mouse; D053590:Interleukin 1 Receptor Antagonist Protein; D017472:Receptors, Interleukin-1; D012795:Sialoglycoproteins