Efficient production of a human tumour necrosis factor in Escherichia coli.
To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D006023:Glycoproteins / Q000235:genetics*; D006131:Growth Inhibitors / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D014176:Protein Biosynthesis; D014409:Tumor Necrosis Factor-alpha
D006023:Glycoproteins; D006131:Growth Inhibitors; D014409:Tumor Necrosis Factor-alpha; D004262:DNA Restriction Enzymes