Bioengineering Recombinant Diacylglycerol Acyltransferases

Abstract

The complete genomes of many organisms including human, mouse, Arabidopsis, and rice have been sequenced. However, the functions of the proteins encoded by a large percentage of the genes in these organisms have not been determined. The immediate challenge of the post-genomic biology is to determine the biological functions of proteins coded for by those unknown genes. Many endogenous proteins occur in extremely low abundance (such as the anti-inflammatory protein tristetraprolin, TTP) (Cao et al., 2004) and are labile (such as omega-3 fatty-acid desaturase, FAD3) (O'Quin et al., 2010), which are major problems inherent to characterization of those proteins. Recombinant proteins can be used as an alternative source to endogenous proteins. Production of active proteins in large quantities is necessary for the study of protein structure and function (Cao et al., 2003). Purified recombinant proteins are also important for the production of antibodies (Cao 2004; Cao et al., 2008; Cao et al., 2004) and pharmaceutical reagents. Unfortunately, a great number of proteins are difficult to express and purify. Those proteins include membrane proteins, lipid-associated proteins, and lowabundance proteins. The causes of the difficulties in protein expression and purification are various, among which are protein insolubility, protein degradation, and low-level protein expression (Cao 2010). Therefore, production of high-quality recombinant proteins requires optimization of protein expression and purification procedures in each case. Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been isolated from many organisms. At least two forms of DGATs are present in mammals (Cases et al., 1998; Cases et al., 2001) and plants (Lardizabal et al., 2001; Shockey et al., 2006) with additional forms reported in burning bush (Euonymus alatus) (Durrett et al., 2010), peanut (Saha et al., 2006), and Arabidopsis (Rani et al., 2010). Plants and animals deficient in DGATs accumulate less TAG (Smith et al., 2000; Stone et al., 2004; Zou et al., 1999). Animals with reduced DGAT activity are resistant to diet-induced obesity (Chen et al., 2004; Smith et al., 2000) and lack milk production (Smith et al., 2000). Over-expression of DGAT enzymes increases TAG content in plants (Andrianov et al., 2010; Bouvier-Nave et al., 2000; Burgal et al., 2008; Durrett et al., 2010; Jako et al., 2001; Lardizabal et al., 2008; Xu et al., 2008), animals (Kamisaka et al., 2010; Liu et al., 2009; Liu et al., 2007; Roorda et al., 2005), and yeast (Kamisaka et al., 2007). DGATs have nonredundant functions in TAG biosynthesis in species