Expression of biologically active human tumor necrosis factor beta by Streptomyces lividans.
The production of human tumor necrosis factor beta (hTNF beta) in Streptomyces lividans was studied. The expression of hTNF beta uses the transcription, translation and secretion signals of subtilisin inhibitor VSI which is naturally produced by Streptomyces venezuelae CBS762.70. In direct secretory expression cassette, hTNF beta cDNA was fused 2 amino acids after the signal peptidase cleavage site. In fusion expression cassette, hTNF beta cDNA was fused after the total vsi gene. In intracellular expression cassette, hTNF beta cDNA was fused after initiation codon ATG. The expression cassettes were subcloned into Streptomyces multi-copy plasmid pIJ486 respectively and transformed into S. lividans TK24. The recombinant strains were designated as S. lividans (pIJ486-hTNF beta), S. lividans (pIJ486-vsi-hTNF beta) and S. lividans (pIVPA-hTNF beta). The analysis of expressed proteins by Western blotting and biological activity measurements revealed hTNF beta was expressed by the recombinant strains with bioactivity. The molecular weight of directly secreted product is around 16 kDa, and the expression level at 48 hours in NB medium was 0.7 mg/L. Intact product could be obtained when hTNF beta was expressed intracellularly, although it may be degraded to lower molecular weight product when cultivation was prolonged. The intracellular expression level at 48 hours in NB medium was 25.1 mg/L.
Hong
Bin
B
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. binhong@95777.com
Li
Yuan
Y
Van Mellaert
Godelieve
G
Anné
Jozef
J
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008233:Lymphotoxin-alpha / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics*
D001426:Bacterial Proteins; D008233:Lymphotoxin-alpha; D011993:Recombinant Fusion Proteins; C116044:subtilisin inhibitor protein VSI, Streptomyces venezuelae