Overexpression of lymphotoxin in escherichia coli and in vitro refolding of insoluble aggregate to biologically active form

Abstract

Overexpression of biologicall active lymphotoxin, i.e. tumor necrosis factor-$\beta$, was attempted using genetic engineering strates. The lympnotoxin gene contained in pLTl was transfered is pASI and constructed a new plasmid pALI. The signal sequence of precursor lymphotoxin gene in pALI was removed. With this pALIII, Escherichia coli M5248 was transformed and production of lymphotoxin was attempted. However, the E. coli transformant having pALIII did not produce lymphotoxin. The $P_L$ promoter of the lymphotoxin gene in pALIII was replaced with trc promoter and constructed prtcLT which produced lymphotoxin below 1\% of total cell proteins in E. coli. To increase the lymphotoxin production, the cDNA sequence of lymphotoxin gene was mutated by altering the translation initiation region using chemically synthesized oligonucleotides avoiding the local secondary structure of mRNA. The mutations were so designed to maintain the original amino acid sequence of lymphotoxin unchanged. The structures of mRNA of lymphotoxin were predicted in the translation initiation region based on the shorth range interaction of mRNA spanning 5`` terminal 130 nucleotides. The expressions of lymphotoxin were increased as the predicted local secondary structure of mRNA decreased. The elevation of cultivation temperature also increased the expression level of lymphotoxin. The amounts of mRNA were not significantly different when the cultivation temperature decreased to 32$^\circ$C or increased to $42^\circ$C. The maximal yield of expression level of lymphotoxin was 12\% of total cell proteins. Most of produced lymphotoxin in this system were biologically active form. Another trial of overproduction was performed by expressing the cDNA sequence of human lymphotoxin gene in T7 expression system. A production of lymphotoxin at the level of 16\% of total cell proteins was observed, but the production was in the form of insoltions by the criteria descrived in above section were resulted in ...