Production of Specific Protease
Production and Specific Properties of IgA Protease Secreted by Neisseria Meningitidis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Evstyukhin K N
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Tufts University / Tufts New England Center Hospital, USA, MA, Boston\nHauptman-Woodward Medical Research Institute, Inc., USA, NY, Buffalo\nUniversity of Aarthus, Denmark, Aarhus
Project summaryImmunoglobulin A (Ig A) is one of the five classes of antibodies, induced in response to the appearance of foreign substances, antigens, in an organism. IgA protect mucosas from adhesion and invasion of pathogenic microorganisms.
IgA are generally resistant to the effect of protheolytic enzymes. However, it has been found recently that some parasites secrete the highly-spec. proteases, which break bonds in in human IgA, (each brealing only one bond). IgA loses thereby its protective properties; and the cell of the parasite gets the opportunity to adhere and reproduce in mucosas. The information about Virulent strains of such microorganisms, for example N.meningitidis differ from nonvirulent ones by hegira higher concentration of the secreted proteases. The study of such proteases, structure and properties of which is still little, can make it possible to find new approaches to the treatment of the diseases, caused by these microorganisms. For example, synthetic peptides, which have the sequence of amino acids similar to the region of cleavage in IgA, could be effective inhibitors of IgA-protease.
The test-systems, developed on the basis of IgA-protease, could help to differentiate virulent and nonvirulent strains.
The Objective of the project
The major objective of the project proposed is:
- to obtain IgA-protease, secreted by virulent N.meningitidis strain and to study its properties.
It is important both for the control over the dangerous disease and for solution of basic problem - the cause of exclusive specification of IgA-proteases. The choice of the object is determined by the importance to control meningitis.
Main research tasks:
- to optimize cultivation of the virulent N.meningitidis strain;
- to isolate and purify the secreted IgA-protease;
- to study physic-chemical properties of the enzyme;
- to predict the structure of peptides and to synthesize them as potential inhibitors or substrates of IgA-protease;
- to develop an immunoenzymatic test-system to determine IgA-protease;
- to obtain and use monoclonal antibodies to IgA-protease;
- to isolate and purify the substrate for this enzyme-monoclonal IgA from sera of patients.
The expected results
The studies, carried out under the project, should facilitate the understanding of molecular bases of exclusive specificity of IgA-proteases, which make them different from serineproteolytic enzymes of chymotrypsin type.
The immunoenzyme systems on the basis of IgA-protease could help to test diagnosis of meningitis in patients and carriers. Production of synthetic inhibitors of the enzyme is a possible way to design an effective medicinal compound of the new type.
Technical Approach and Methodology
There is a good methodological basis for the execution of the project at the State Scientific Center of Applied Microbiology. Thus, employees of the Center use in their studies the method of computer simulation of protein steric structure, synthesis of the peptides, having biological activity, procedures to obtain and purify monoclonal antibodies and physic-chemical methods to study protein properties, methods of development of test-systems and isolation and purification of proteins.
A range of physico-chemical methods will be used:
- spectrophotometric methods;
- the method of chemical modification of ammo acid residues;
- adiabatic scanning difference microcalorimetry;
- determination of specificity, etc.
The pathogenic N.meningitidis strain is supposed to be cultivated at the State Scientific Center of Applied Microbiology experienced in cultivation of pathogenic microorganisms. Thus we have a good methodological base for every stage. Besides, it should be noted that the participants of this project have broad experience in investigating properties of proteolytic enzymes.
International cooperation under the project submitted to the ISTC. It is planned to exchange the information received under the project, to prepare joint publications, to exchange samples required for research, to make visits to laboratories of collaborators aiming at carrying out the work under the project, to organize scientific seminars at the State Scientific Center of Applied Microbiology with the participation of collaborators.
This project has positive estimation from Dr. A. Wlodamer (USA) and Prof. D' Ari (France). They will take part in proposed project.