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Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F
Abstract:
The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes.
Insights
The Pseudomonas aeruginosa porin protein F gene was cloned into E. coli, successfully expressing functional porin F. This study confirms the gene
Area of Science:
- Microbiology
- Molecular Biology
- Biophysics
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen.
- Porins are essential outer membrane proteins involved in nutrient transport.
- Understanding porin function is crucial for developing antimicrobial strategies.
Purpose of the Study:
- To clone and express the gene for porin protein F from Pseudomonas aeruginosa in Escherichia coli.
- To characterize the expressed protein F and confirm its identity and function.
- To investigate the functional properties of cloned porin F in artificial membranes.
Main Methods:
- Gene cloning using a cosmid vector into E. coli.
- Outer membrane protein analysis via SDS-PAGE.
- Protein characterization using 2-mercaptoethanol modification and monoclonal antibody reactivity.
- Southern blot hybridization for gene copy number determination.
- Purification of expressed porin F.
- Reconstitution of purified porin F into black lipid bilayer membranes for channel activity analysis.
Main Results:
- Porin protein F was successfully expressed as a predominant outer membrane protein in a porin-deficient E. coli.
- Expressed protein F showed identical characteristics to native P. aeruginosa porin F.
- A truncated protein F derivative was produced from a subcloned fragment.
- Southern blots indicated a single copy of the porin F gene in P. aeruginosa.
- Reconstituted porin F formed channels with conductances similar to native porin F.
Conclusions:
- The gene for Pseudomonas aeruginosa porin protein F can be functionally expressed in E. coli.
- Cloned porin F retains its structural and functional properties, including channel formation.
- This work provides a basis for further studies on porin structure-function relationships and potential therapeutic targets.