The expression of human tumor necrosis factor in E. coli.
cDNA of human natural TNF (n-TNF) obtained by stimulating human leukemic B cell line (Ball-1) with Sendai virus was cloned. Valine-started-TNF (V-TNF) gene was constructed from the cDNA and expressed in E.coli HB101 under the control of a trp promoter by the induction of 3-indoleacrylic acid. The expression level of V-TNF clone was about 10% of the total E.coli protein. On the other hand, the expression level of glutamine started-TNF (Q-TNF) gene having the same SD-ATG sequence which was constructed from V-TNF gene was as low as about 1/20 of that of V-TNF. The nucleotide sequence around ATG (-4 approximately +12) of Q-TNF gene was randomly changed without modifying the coded amino acid sequence, resulting to obtain high expression clones as similar TNF protein yield as that of V-TNF. These clones possessed A residue rich sequence around the initiation codon ATG. These results show that some correlation might exist between the high expression rate and A residue rich sequence around the initiation codon.
D000595:Amino Acid Sequence; D001402:B-Lymphocytes; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D010222:Parainfluenza Virus 1, Human / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha
D006023:Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA