Expression, biological activity and kinetics of production of recombinant ovine TNF-alpha.
Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 degrees C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-alpha from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L-1. Both rOvTNF-alpha and recombinant human TNF-alpha (rhTNF-alpha) exerted cytotoxicity on L929 cells. However, rOvTNF-alpha but not rhTNF-alpha stimulated proliferation of ovine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.
Seow
H F
HF
CSIRO Division of Animal Health, Animal Health Research Laboratory, Parkville, Vic., Australia.
Rothel
J S
JS
Pepin
M
M
David
M J
MJ
Wood
P R
PR
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008213:Lymphocyte Activation / Q000276:immunology; D008262:Macrophage Activation / Q000276:immunology; D016676:Macrophages, Alveolar / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012756:Sheep / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D012313:RNA