International network for natural sciences – research journal
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Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in Escherichia coli

By: Meghana Kolavalli Jayanth, Paramanahally Hanumanthe Gowda Ramanjini Gowda, Neha Guleria, Satish Kumar Kariyaiah

Key Words: Diabetes mellitus, GAD65, SDS-PAGE, Western blot.

Int. J. Biomol. & Biomed. 5(2), 1-8, August 2016.

Abstract

Diabetes is a chronic autoimmune disease characterized by the inability of body to produce or respond to insulin a hormone required by body to burn glucose for energy. Type I Diabetes mellitus, also known as Insulin Dependent Diabetes mellitus is a most frequent chronic disease of childhood, afflicts 0.2-0.3% of human individuals due to auto immune destruction of insulin secreting pancreatic β cells. GAD65 is the major auto antigen in Insulin Dependent Diabetes Mellitus (IIDM). Thus, this project is aimed at expression of GAD65 in E. coli. GAD65 gene was cloned into pET-28a bacterial expression vector and expression was studied in BL21 DE3 cells. Different parameters of induction like isopropyl-β-D-thiogalactopyranoside (IPTG), temperature, time interval were standardized. The recombinant clones induced with 2 μM of IPTG at 30oC for 4 h at flask level produced the protein upto 537μg/ml. Furthermore, the specificity of the purified recombinant protein was confirmed by western blot analysis using monoclonal antibodies. This work establishes a strategy in E. coli for the expression of GAD65 with optimized parameters.

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