1. Field Of The Invention
The present invention relates to the field of recombinant DNA technology and to the use of araB promoters in the expression of heterologous genes in transformed hosts. This patent also relates to the design, cloning and expression of genes coding for the bactericidal peptide cecropin and analogues thereof.
2. Brief Description of the Background Art
Genetic information, encoded in DNA molecules, is expressed by a series of steps involving transcription of the DNA into mRNA and the subsequent translation of the mRNA into polypeptides or proteins. The expression of the encoded information to form polypeptides is initiated at the promoter site, a region on the DNA molecule to which RNA polymerase binds and initiates transcription. Promoters that have been used in recombinant DNA methods for expressing heterologous genes include the beta-lactamase (penicillinase) and lactose (beta-galactosidase) promoter systems (Chang et al., Nature, 275:615 (1978); Itakura et al., Science, 198: 1056 (1977); Goeddel et al., Nature, 281:544 (1979)) and tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 8:4057 (1980); EPO Application Publication No. 0036776). Other known promoters include the bacteriophage lambda promoters, (P.sub.L) and (P.sub.R), hut, colicin E.sub.1, galactose, alkaline phosphatase, xylose A, and tac.
The araB gene and its promoter (araB) are located in the L-arabinose operon. The L-arabinose operon (araBAD) in Escherichia coli and in Salmonella typhimurium has been studied. Of particular interest is the L-arabinose operon in S. typhimurium; its sequence is described in Horwitz, A. et al., "DNA Sequence of the araBAD-araC Controlling Region in Salmonella typhimurium LT2," Gene, 14: 309-319 (1981), Lin, H.-C. et al., "The araBAD Operon of Samonella Typhimurium LT2, I. Nucleotide Sequence of araB and Primary Stucture of Its Product, Ribulokinase," Gene, 34:111-122 (1985); II. "Nucleotide Sequence of araA and Primary Structure of Its Product, L-Arabinose Isomerase," Gene, 34:123-128 (1985); III. "Nucleotide Sequence of araD and Its Flanking Regions, and Primary Structure of its Product, L-Ribulose-5-Phosphate-4-Epimerase," Gene, 34:129-134 (1985). The araBAD operon contains three structural genes which are responsible for the initial metabolism of L-arabinose. Lee, Jar-How et al., "Genetic Characterization of Salmonella typhimurium LT2 ara Mutations," J. of Bacteriology, 158:344-46 (1984). L-arabinose is first converted into L-ribulose by the araA gene product, L-arabinose isomerase. L-ribulose is then phosphorylated to L-ribulose-5-phosphate by the araB gene product, ribulokinase. The araD gene product. L-ribulose-5-phosphate 4-epimerase, catalyzes the conversion of L-ribulose-5-phosphate to D-xylose-5-phosphate which then enters the pentose phosphate pathway. The araBAD operon is coordinately controlled by the inducer L-arabinose and the araC regulatory gene product.
Since, in one embodiment of the invention disclosed and claimed herein, the araB promoter is operably linked to the gene coding for cecropin and inserted into a suitable host for expression of the cecropin protein, it is worthwhile to review background references on cecropins.
The immune system of the Cecropia moth and several lepidopteran insects is characterized by an effective humoral response which is mainly associated with the cecropins, a recently discovered family of antibacterial peptides (Boman, H. G. and Steiner, H., Current Topics In Microbiology And Immunology, 94/95:75-91 (1981)). Three major cecropins, A, B and D, have been purified from immune hemolymph and their sequences have been elucidated (Steiner, et al., Nature. 292: 246-248 (1981); Qu, et al., European Journal of Biochemistry, 127:219-224 (1982); Hultmark, D., ibid, 127:207-217 (1982); and Hultmark, U.S. Pat. No. 4,355,104). All cecropins are small basic peptides with a high degree of mutual sequence homology. The amino acid sequences of cecropins B and D from Antheraea pernyi (A.p.) and from Hylophora cecropia (H.c.) are as follows: ##STR1##
The cecropins are similar in structure to the bee venom toxin melittin, but have a broader antibacterial spectrum than mellitin, and do not lyse cultured liver cells, sheep erythrocytes or insect cells. As shown above, the carboxy terminus in all cecropins is blocked and, in the case of cecropin A, the blocking group is a primary amide (Andreu, et al., Proceedings of the National Academy of Sciences, USA, 80:6475-6479 (1983)). Cecropin A and several related peptides have recently been synthesized by solid phase techniques and have been shown to be totally indistinguishable from natural cecropin A by chemical and physical criteria (Andreu, et al., supra).
Interestingly, the carboxy terminal tetrapeptide imide was found to be of little importance for the antibacterial activity towards E. coli, but for three other bacteria tested, the activity was reduced to 3% to 20% of that of cecropin A.
The cecropins are antibacterial against a variety of bacteria including both Gram-negative and Gram-positive bacteria. The available data on the mode of action of the cecropins indicate that they disrupt the cytoplasmic membranes of bacteria (Steiner, et al., Nature, 292:246-248 (1981)). It is apparent from the literature that different bacterial species have different sensitivities to the cecropins, and that each cecropin has a distinct spectrum of activity. For example, Bacillus megaterium is highly sensitive to cecropins A and B, but relatively resistant to cecropin D. Both Gram-negative and Gram-positive organisms have been shown to be sensitive to cecropins in the micromolar concentration range. Organisms showing a high level of sensitivity to cecropins include E. coli, Pseudomonas aeruginosa, Serratia marsescens, Xenorhabdus nematophilus, B. megatherium, and Micrococcus luteus. Although cecropins A and B show a total of twelve amino acid replacements, their activities against nine different bacterial species are very similar, suggesting that many amino acid substitutions can be tolerated without altering the biological activity of the peptide. Similarly, cecropin B from the Chinese oak silk moth (A. pernyi) differs from cecropin B from North American silk moths (H. cecropia) at four positions; however, three of the changes are replacements for the corresponding amino acids found in the H. cecropia A form. The fourth change is in a position where H. cecropia A and B forms differ and is a conservative change. It is therefore apparent that unique derivatives of the cecropins created by conservative amino acid substitutions would retain their biological activity. Non-conservative changes such as those found in cecropin D might be expected to alter the activity of the peptide. Cecropin D has almost as much activity against E. coli as cecropins A and B, but has significantly reduced activity against eight other species of bacteria.
In view of the great usefulness of the cecropins and analogues thereof and of the great promise that recombinant DNA methods offer for the production of proteins, it appeared desirable to provide a system for the production of cecropins by means of such technology.