Cloning and Expression of Human GAD65 Gene in E. coli | InformativeBD

Meghana Kolavalli Jayanth, Paramanahally Hanumanthe Gowda Ramanjini Gowda, Neha Guleria, and  Satish Kumar Kariyaiah  from the different institute of the india wrote a research  article about  Cloning and Expression of Human GAD65 Gene in E. coli, entitled  " Cloning and expression of Human glutamic acid decarboxylase (GAD 65) gene in Escherichia coli " this research paper published by the International Journal of Biomolecules and Biomedicine (IJBB)  an open access scholarly research journal of Biomedicine under the affiliation of the International Network for Natural Sciences | INNSpub an open access multidisciplinary research journal publisher.

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.

Introduction

Diabetes mellitus is a disease or disorder that is gaining importance and increasing at the alarming rate, which is also called as hyperglycemia where the blood glucose level is higher than the normal. According to World Health Organization, there are mainly two principle types of diabetes; Type 1 diabetes (juvenile or insulin dependent diabetes) and type 2 diabetes (insulin independent diabetes).Type 1diabetes an autoimmune form of diabetic disorder where the insulin producing β cells of the pancreatic islets are destroyed. It is commonly seen in children and the worldwide numbers of prevalent cases of T1D in children (<15 years) have increased and the estimates indicate that there are almost 500,000 children aged under 15 years with T1D worldwide (Patterson et al., 2014). In this type of diabetes, the autoimmune response occurs against self-antigens which include insulin, intracellular membrane proteins such as GAD, IA2, ZnT8 transporter protein (La Torre and Lernmark, 2010; Wenzlau et al., 2009). There is currently no cure for T1D and the only available treatment is insulin therapy. Insulin therapy can lead to hypoglycemia if the doses are not taken properly without monitoring blood glucose level and patients requires two or more injections of insulin daily which is painful. (Alvarez et al., 2013).In this regard, GAD65 a major auto antigen have much potential as an important marker for the prediction, diagnosis and in the prevention of T1D in humans. Autoantibodies to GAD65 are observed months to years before the clinical onset of diabetes and are present in the sera of 70–80% of patients with T1D and this anti-GAD 65 antibodies are now serving as an important marker for the prediction and diagnosis of type 1 diabetes (Jayakrishnan et al., 2011; Oak et al., 2011; Wang et al., 2012).

Glutamic acid decarboxylase is an enzyme which is involved in the production of Gamma amino butyric acid (GABA) by the decarboxylation of glutamate is an inhibitory neurotransmitter in neurons and pancreatic beta cells. This GAD exists in two major protein isoforms.

One isoform has a molecular size of 65kDa and is termed GAD 65, while the second one, of 67kDa size, is termed GAD67 (Towns and Pietropaolo, 2011). The GAD65 enzyme isolation in large quantities from the human pancreatic (beta cells) tissue is unrealistic so the expression of GAD 65 enzyme as a recombinant protein in heterologous system is mandatory. The large scale production of GAD 65 enzyme currently involves the use of baculovirus infected Sf9 insect cells and methylo-trophic yeast (Mauch et al., 1993; Moody et al., 1995). However, these expression system are technically, economically demanding and highly vulnerable to contamination.

Number of heterologous biopharmaceutical proteins expressed in E. coli which are at commercial level are innumerable when compared to all other hosts system this is because of reasons like, it is inexpensive, offers rapid culture times and the ability to achieve high biomass and high protein yields (Assenberg et al., 2013). E. coli expression system remain to be the first choice for laboratory investigations, scaling up activities at commercial level and are useful benchmark for comparison among various expression platforms. The sufficient quantity of soluble and functional recombinant GAD 65 protein is required to carry out any molecular and immunological studies. Poor expression of protein because of cellular toxicity and formation of inclusion bodies are the major hindrance in recombinant protein expression. Therefore, this study was carried out for the optimization of different parameters to enhance GAD 65 protein expression and purification in E. coli.

Source: Cloning and expression of Human glutamic acid decarboxylase(GAD 65) gene in Escherichia coli