Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in Escherichia coli
Paper Details
Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in Escherichia coli
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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By: Meghana Kolavalli Jayanth, Paramanahally Hanumanthe Gowda Ramanjini Gowda, Neha Guleria, Satish Kumar Kariyaiah (2016), Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in Escherichia coli; IJBB, V5, N2, August, P1-8
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