Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli

Biochim Biophys Acta. 1994 May 17;1218(1):11-20. doi: 10.1016/0167-4781(94)90095-7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adhesins, Bacterial*
  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Base Sequence
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cloning, Molecular
  • DNA, Bacterial
  • Escherichia coli / genetics*
  • Genes, Bacterial
  • Hemagglutination Tests
  • Humans
  • Immune Sera
  • Lectins / genetics*
  • Molecular Sequence Data
  • Precipitin Tests
  • Pseudomonas aeruginosa / genetics*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Adhesins, Bacterial
  • Bacterial Proteins
  • DNA, Bacterial
  • Immune Sera
  • Lectins
  • Recombinant Proteins
  • adhesin, Pseudomonas

Associated data

  • GENBANK/X65933