Cloning and expression of recombinant biopharmaceutical L-asparaginase from Escherichia coli with ELP-intein tag

• Grant number: 14/02782-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: May 01, 2014
• End date: April 30, 2015
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Gisele Monteiro
• Grantee: Lucas Fonseca Gonzaga
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Leukemia took first place among the causes of death from cancer in individuals under 18. As a treatment protocol for acute lymphoblastic leukemia (ALL) is used, among other chemotherapeutic, the enzyme L –asparaginase (ASPase), which plays a central role because the amino acid asparagine to deplete induces leukemic lymphoblasts to enter into apoptosis. There are a total of three types of ASPases that are used therapeutically, however, Brazil only uses for treating the native form of Escherichia coli (Elspar ®). Brazil does not produce any industrial biopharmaceutical, and purification procedures one of the most expensive steps in getting these, as a rule involve chromatography. Since the relevance of biopharmaceutical, we propose to construct a vector that allows the overexpression of the enzyme L- asparaginase, and chromatographic steps without further purification. The method comprises adding to the protein of interest from a tail with two areas of particular properties called " ELP- intein". The first field (elastin like polypeptide - PLA) precipitates reversibly with the average temperature rise, allowing the separation of the complex into ELP- intein fusion -asparaginase the fermentation broth or cell lysate by centrifugation. The second domain (intein) performs the spontaneous self - cleavage under changing pH of the solution, releasing the ELP- intein tail asparaginase. Thus, by raising the temperature and centrifuge again, asparaginase will be free and without fusion in the supernatant. This design shows how the proposed construction of a fusion protein that will allow the use of a viable and economical for the production of recombinant asparaginase on an industrial scale in Brazil technique.