Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli.
This communication describes a Pseudomonas aeruginosa DNA fragment (cloned in lambda gt11) which contains the structural gene coding for the galactophilic PA-I lectin (pa-1L, 369 bp) and an additional downstream 237 bp sequence. This DNA is relatively rich in G + C (54%), and exhibits a strong codon preference biased for XXC and also for XXG. The Shine-Dalgarno site of the gene is preceded by an adjacent ATATAT sequence resembling the -10 sequence of the Escherichia coli promoter. The stop codons are followed by a stem and loop structure--typical of the rho-independent transcriptional stop element. This lambda gt11-cloned DNA was expressed in E. coli Y1090 cells. The resulting cell lysates exhibited a galactose-specific hemagglutination and a protein with electrophoretic 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 affinity chromatography, was shown to be a galactophilic hemagglutinin resembling the native lectin in molecular weight and selective reactivity with rabbit anti native PA-I serum. These results are important for development of a safe Pseudomonas aeruginosa vaccine using recombinant DNA techniques, thus avoiding contamination with toxic products of this bacterium.
Avichezer
D
D
Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Gilboa-Garber
N
N
Garber
N C
NC
Katcoff
D J
DJ
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006386:Hemagglutination Tests; D006801:Humans; D007106:Immune Sera; D037102:Lectins / Q000235:genetics*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007106:Immune Sera; D037102:Lectins; D011994:Recombinant Proteins; C038692:adhesin, Pseudomonas