SYNTHESIS AND EXPRESSION OF A GENE FOR HUMAN TUMOR NECROSIS FACTOR-ALPHA (TNF-α)

TNF-α was found originally In sera of Bacillus Calmette Guerln infected mice as a macrophage derived factor. It Is cytotoxlc for tumor cell and less or not toxic to normal cells in vitor. The gene for human TNF-α with E. coli-preferred codons has been designed according to the amino acid sequence deduced from the cDNA. The gene with 504 bp was divided into 27 oligonucleotide fragments having 30. to 40 nucleotides each. The solid phase phosphotriester method was used for the synthesis of these oligonucleotides. The 27 fragments were annealed to three segments and then linked by T4 DNA ligase. The entire gene was incorporated into plasmld PDR540 with Tac promoter which was used to transform E. coli 7118. The expressed protein was estimated by SDSPAGE with a molecular weight of 1. 7×104Da. The cytotoxlc activity of the product against L-929 cell was 1. 0×107units/ml culture.

Synthesis and expression of a gene for human tumor necrosis factor-alpha (TNF-α)

TNF-α was found originally in sera of Bacillus Calmette Guerin infected mice as a macrophage derived factor. It is cytotoxic for tumor cell and less or not toxic to normal cellsin vivo. The gene for human TNF-α withE. coli-preferred codons has been designed according to the amino acid sequence deduced from the cDNA. The gene with 504 bp was divided into 27 oligonucleotide fragments having 30 to 40 nucleotides each. The solid phase phosphotriester method was used for the synthesis of these oligonucleotides. The 27 fragments were annealed to three segments and then linked by T4 DNA ligase. The entire gene was incorporated into plasmid PDR540 with Tac promoter which was used to transformE. coli 7118. The expressed protein was estimated by SDSPAGE with a molecular weight of 1.7×10⁴ Da. The cytotoxic activity of the product against L-929 cell was 1.0×10⁷ units/ml culture.