PSEUDOMONAS-AERUGINOSA PA-I LECTIN GENE MOLECULAR ANALYSIS AND EXPRESSION IN ESCHERICHIA-COLI

This communication describes a Pseudomonas aeruginosa DNA fragment (cl oned in lambda gt11) which contains the structural gene coding for the galactophilic PA-I lectin (pa-1L, 369 bp) and an additional downstrea m 237 bp sequence. This DNA is relatively rich in G + C (54%), and exh ibits a strong codon preference biased for XXC and also for XXG. The S hine-Dalgarno site of the gene is preceded by an adjacent ATATAT seque nce resembling the - 10 sequence of the Escherichia coli promoter. The stop codons are followed by a stem and loop structure - typical of th e rho-independent transcriptional stop element. This lambda gt11-clone d DNA was expressed in E. coli Y1090 cells. The resulting cell lysates exhibited a galactose-specific hemagglutination and a protein with el ectrophoretic mobility similar to that of the native PA-I, which were both absent from E. coli lysates infected with ovalbumin gene-bearing bacteriophages. The recombinant PA-I, purified by gel filtration and a ffinity chromatography, was shown to be a galactophilic hemagglutinin resembling the native lectin in molecular weight and selective reactiv ity with rabbit anti native PA-I serum. These results are important fo r development of a safe Pseudomonas aeruginosa vaccine using recombina nt DNA techniques, thus avoiding contamination with toxic products of this bacterium.