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Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia
M Duchêne1, C Barron, A Schweizer
1Laboratorium für Molekulare Biologie, Ludwig-Maximilians-Universität München, Federal Republic of Germany.
Abstract:
Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections.
Insights
Lipoprotein I (OprI) from Pseudomonas aeruginosa is a potential vaccine candidate. Researchers successfully isolated and expressed the OprI gene in E. coli for further testing against infections.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Lipoprotein I (OprI) is a major outer membrane protein of Pseudomonas aeruginosa.
- OprI, similar to porin protein F (OprF), shows antigenic cross-reactivity with all serotype strains, making it a vaccine candidate.
Purpose of the Study:
- To isolate the gene encoding Lipoprotein I (OprI) from Pseudomonas aeruginosa.
- To express OprI in Escherichia coli for further study and potential vaccine development.
Main Methods:
- Screening a lambda EMBL3 phage library using a mouse monoclonal antibody against OprI.
- Gene isolation and expression of OprI in E. coli.
Main Results:
- The OprI gene was successfully isolated and expressed in E. coli.
- The OprI mRNA is monocistronic, encoding an 83-amino acid precursor with a 19-residue signal peptide.
- The mature OprI protein has a molecular weight of 6,950 (excluding bound glycerol and lipid).
Conclusions:
- The successful expression of OprI in E. coli allows for testing its protective effect against P. aeruginosa infections without lipopolysaccharide contamination.
- This advancement facilitates the evaluation of OprI as a standalone vaccine component.