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Expression of cysteine protease rCPB2.8 of Leishmania mexicana
Brazil (IFMSA Brazil) - Universidade de Mogi das Cruzes (UMC), Mogi das Cruzes.
Centro Interdisciplinar de Investigacao Bioquimica-CIIB 200 Candido Xavier de Almeida e Souza Av. Vila Partenio, ZIPCODE:08780-911
Wagner Alves de Souza Judice, PhD
Wagner Alves de Souza Judice, PhD
Type of Research Project
- Basic science
What is the background of the project?
The project involves transforming E. coli bacteria with a cysteine protease enzyme expression plasmid rCPB2.8 that is involved in the process of infectivity and virulence in leishmaniasis disease that affects millions of individuals in underdeveloped countries. The plasmid incorporated into the bacterium confers resistance to the antibiotic ampicillin allowing selection of the clone carrying the expression vector of the enzyme. The clone containing the expression vector will be cultured in LB liquid medium and the protein expression will be induced by IPTG. The protein expressed in the liquid medium will be purified by Nickel-Sepharose Resin Affinity Chromatography and the retained material will be eluted in an imidazole concentration gradient. The enzymatic activity was monitored by the hydrolysis of the fluorogenic substrate Z-FR-MCA by spectrofluorimetry using 100 mM sodium acetate buffer, pH 5.5 at 37 ° C.
What is the aim of the project?
Identify molecules derivatives from morpholinics eugenol as modulates of rCPB2.8 cysteine protease from L. mexicana. The purified enzyme will be used as a tool for the development of possible leishmanicidal drugs.
What techniques and methods are used?
Molecular biology, spectrofluorimetry, molecular cloning, enzyme kinetics Speectrofluorometry to identify the cleavage of fluorogenic substrates as probe associated at water bath to stabilize the temperature of kinetic reaction in both situation, in the absence and in the presence of modulator molecule as possible inhibitor. The results will be categorized and analyzed threw Excel tables.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The beginning of internship the student he/she will observed the procedures performed by master or PhD student and after will performed the experimental procedures by yourself assisted by the higher student.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Will be provide readings and lectures to student.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student’s name will be mentioned in a future publication
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
There no required skills but the student should be dedicated to the project. Subjects passed: Biochemistry
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Pre-Medical students from the American-British system
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- Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.
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