Ping Li (2012-2013)
Assistant Professor, Department of Chemistry
Kansas State University
Expression and purification of an obesity-important enzyme hGOAT in E.coli
Ghrelin O-acyltransferase (GOAT) is the only enzyme responsible for the formation of biologically active acylated ghrelin that has been linked to obesity. Sufficient amount of human GOAT (hGOAT) is a prerequisite to study its molecular mechanisms, which will lead to new treatment for obesity. Development of expressing and purifying hGOAT heterologously in E. coli will ensure that enough enzymes can be obtained.
Ghrelin is a peptide hormone that consists of 28 amino acids and regulates our appetite and adiposity in humans. The active acylated ghrelin that has an octanoyl group attached to Ser-3 will increase the appetite and slow down metabolism, causing the body to feel hungrier and to burn fat slowly, thus predisposing the person to obesity. The formation of acylated ghrelin is catalyzed by ghrelin O-acyltransferase (GOAT), which was discovered recently in 2008. The US is currently facing obesity pandemic, which costs more than 150 billion dollars per year.
Obesity has become a major contributing factor in human deaths from diabetes, cancers, and cardiovascular diseases. Therefore, it is more urgent than ever for us to develop effective ways to treat obesity. Mouse model has suggested that inhibition of GOAT could provide promising therapeutics for treating obesity. However, studies of human GOAT (hGOAT) are hampered due to the insufficient amount of pure enzymes that can be obtained.
The goal of this pilot project is to express and purify membrane protein hGOAT heterologously from Escherichia coli. The specific aims are: (1) To develop protocols for the overexpression of hGOAT in E. coli with high yields. Strategies will be developed on how to design constructs and to choose the appropriate expression promoters and host strains. In order to increase the expression levels of soluble hGOAT, focus will be placed on the design of fusion proteins and the reasons why they are selected are described; and (2) To develop protocols for the renaturation of hGOAT from inclusion bodies. Two methods will be employed to obtain the soluble active hGOAT from its inclusion bodies. Different detergents will also be tested for extracting hGOAT and maintaining its activity. Although this project will involve lots of trials and errors, we believe, by working closely with the experts from COBRE facilities (Protein Production Group and Protein Structure Laboratory at KU), it will lead to obtain sufficient amount of active hGOAT in soluble form from the E. coli recombinant expression system.