[Gene cloning and expression of TNF in Streptomyces lividans TK54]

With the plasmid pIJ486 as a vector, TNF cDNA originated from plasmid pHT1 has been cloned into Streptomyces lividans TK54. After transformation, hundreds of transformants, resistant to neomycin (30 micrograms/ml) were selected. Result showed that TNF cDNA was inserted into the plasmid pIJT7 harboured in No. 7 transformant, namely Streptomyces lividans TK54-HT. Cytotoxic activity experiment of L929 cell demonstrated that the maximal TNF activity in Streptomyces lividans TK54-HT is more than 10(8) unit/L culture. The neutralization experiment showed that the product producing by Streptomyces lividans TK54-HT is human TNF-alpha. SDS-PAGE confirmed that there is a 17,000 dolton protein band which has the same molecular weight as that of TNF in the clear lysate supernatant of Streptomyces lividans TK54-HT cells. All of these results mentioned above showed that the TNF gene has been successfully cloned and expressed in Streptomyces lividans TK54.