Hong Bin, Li Yuan, Van Mellaert Godelieve, Anné Jozef
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Yi Chuan Xue Bao. 2003 Mar;30(3):209-14.
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.
研究了在变铅青链霉菌中生产人肿瘤坏死因子β(hTNFβ)的情况。hTNFβ的表达利用了委内瑞拉链霉菌CBS762.70天然产生的枯草杆菌蛋白酶抑制剂VSI的转录、翻译和分泌信号。在直接分泌表达盒中,hTNFβ cDNA在信号肽酶切割位点后2个氨基酸处融合。在融合表达盒中,hTNFβ cDNA在整个vsi基因之后融合。在细胞内表达盒中,hTNFβ cDNA在起始密码子ATG之后融合。这些表达盒分别亚克隆到链霉菌多拷贝质粒pIJ486中,并转化到变铅青链霉菌TK24中。重组菌株分别命名为变铅青链霉菌(pIJ486 - hTNFβ)、变铅青链霉菌(pIJ486 - vsi - hTNFβ)和变铅青链霉菌(pIVPA - hTNFβ)。通过蛋白质免疫印迹分析表达的蛋白质和生物活性测量表明,重组菌株表达了具有生物活性的hTNFβ。直接分泌产物的分子量约为16 kDa,在NB培养基中48小时的表达水平为0.7 mg/L。当hTNFβ在细胞内表达时可以获得完整产物,尽管培养时间延长时它可能会降解为较低分子量的产物。在NB培养基中48小时的细胞内表达水平为25.1 mg/L。