Amino acid sequence of the signal peptide of ompA protein, a major outer membrane protein of Escherichia coli.

Purpose: To evaluate prokaryotic expression of the Escherichia coli (E. coli) outer membrane protein A (OmpA) and its immunoprotective function against the main pathogens of animal mastitis.Methods: A molecular cloning method was used to develop a prokaryotic strain expressing OmpA protein, which was purified by Ni-affinity chromatography. Polyclonal antiserum was generated in mice immunized with OmpA protein. Enzyme-linked immunosorbent assay (ELISA) and western blotting were used to determine the titer and verify anti-OmpA serum specificity, respectively. Interaction between OmpA antiserum and main pathogens of animal mastitis was verified by ELISA and a pull-down method. The immune protective function of OmpA protein was evaluated in mice challenged with pathogens of animal mastitis. Optimal fermentation conditions to produce OmpA protein were determined by the L9(34) orthogonal test.Results: A prokaryotic strain expressing OmpA protein was developed, and purified OmpA was used to develop a mouse polyclonal antibody. The anti-OmpA serum exhibited high specificity and a titer of 1:1600. Anti-OmpA serum directly interacted with E. coli and Staphylococcus aureus (S. aureus). OmpA demonstrated a significant immune protective function of 58.33 % against E. coli and 46.15 % against S. aureus. The optimal conditions for expressing fermentation OmpA were a strain absorbance of 0.5 at a wavelength of 600 nm, IPTG final concentration of 0.3 mmol/L, induction time of 12 h, and induction temperature of 28 °C.Conclusion: OmpA possesses selective immunogenicity and a significant immune protective effect against the main pathogens of animal mastitis. The results suggest that OmpA may potentially be used as a vaccine for animal mastitis. Keywords: E. coli, OmpA protein, Immunoprotection, Animal mastitis, Protein fermentation

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Sequence of the regulatory region of ompT, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: Implications for regulation and processing

MGG Molecular & General Genetics ◽

10.1007/bf00382077 ◽

1984 ◽

Vol 193 (3) ◽

pp. 414-421 ◽

Cited By ~ 3

Author(s):

Gerald Gordon ◽

Randall C. Gayda ◽

Alvin Markovitz

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein A ◽

Regulatory Region ◽

Major Outer Membrane Protein ◽

Outer Membrane Protein A ◽

K 12

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Molecular Cloning, Sequencing, and Expression ofomp-40, the Gene Coding for the Major Outer Membrane Protein from the Acidophilic Bacterium Thiobacillus ferrooxidans

Applied and Environmental Microbiology ◽

10.1128/aem.66.6.2318-2324.2000 ◽

2000 ◽

Vol 66 (6) ◽

pp. 2318-2324 ◽

Cited By ~ 37

Author(s):

Nicolas Guiliani ◽

Carlos A. Jerez

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Amino Acid Sequence ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Thiobacillus Ferrooxidans ◽

Sodium Dodecyl ◽

Major Outer Membrane Protein ◽

Gene Coding ◽

Chemolithoautotrophic Bacteria

ABSTRACT Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterizedomp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.

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