Cloning and expression of recombinant bovine glycine N-acyltransferase from bovine liver

Abstract

The importance and social focus on detoxification and improving a person’s lifestyle has increased tremendously over the years. This is due to increasing levels of pollution and the resulting increase of foreign chemicals (toxins) introduced into our bodies. Detoxification is the process of clearing toxins from the body by turning them into other components that are not harmful to the body. Detoxification has become a popular topic and it is well established to the point where detoxification profiling is becoming available as a commercial service. Detoxification is a multiple reaction system that is divided into four phases: Phase 0, Phase I (Functionalization), Phase II (Conjugation) and Phase III (Elimination). The focus of this MSc project was on glycine N-acyltransferase (GLYAT) which is a transferase enzyme involved in Phase II detoxification, specifically glycine conjugation. GLYAT is responsible to detoxify benzoic acid, a xenobiotic widely used in food preservatives to form a glycine conjugate known as hippuric acid (benzoyl glycine). The GLYAT conjugation reaction is also important for the metabolism of drugs such as Aspirin (Levy, 1965). More important for this study, is the fact that glycine supplementation is commonly used in the treatment of specific inborn errors of metabolism (IEM). When some metabolic pathways are defective, accumulating substrates that form can conjugate with glycine and excreted, thereby lessening toxicity in the body. Although humans are closer to mice than bovine on a genetic level, bovine and humans have sufficient DNA sequence similarity to map the human genome almost entirely to the genome of the bovine. In this study, the differences and similarities of the molecular characteristics of bovine GLYAT (bGLYAT) and the human family of GLYAT enzymes were compared. The goal of this study was to clone bGLYAT and generate a recombinant bGLYAT. The recombinant enzyme can then be used for further studies to compare the enzymatic characteristics and species specificity of human GLYAT to bovine GLYAT. I used bacteria to express bGLYAT. Bacteria are simple organisms and lack the ability to fold some recombinant eukaryotic proteins correctly. Molecular chaperones have been demonstrated to be involved in protein folding. Co-expression of chaperones such as trigger factor, GroEL-GroES and DnaK-DnaJ-GrpE with the target protein in bacteria may enable efficient folding of expressed proteins and enhances the production of active enzymes. In Chapter 2 I described how I investigated whether the chaperone trigger factor (TF) can assist with protein folding of recombinant bGLYAT. I prepared cDNA from bovine liver total RNA. The open reading frame (ORF) of bGLYAT was amplified using gene specific primers with addition of NdeI and SalI sites in a two step RT-PCR procedure. bGLYAT was cloned into pColdTF and sequenced. The plasmid containing bGLYAT in the correct reading frame was then introduced into Origami™ cells. pColdTF is a vector that expresses trigger factor (56 kDa) as a fusion protein with recombinant proteins. bGLYAT with fusion protein (88 kDa) was over-expressed and was soluble. However, the recombinant bGLYAT expressed as a fusion protein with TF did not have any enzyme activity. It was speculated that the fusion protein might be affecting the enzyme activity. After removal of the fusion protein by means of thrombin cleavage, bGLYAT (36 kDa) still did not have any enzyme activity. In Chapter 3 I described how I investigated whether bGLYAT (without any fusion proteins) co-expressed with chaperones would be soluble and enzymatically active. Sequencing results confirmed cloning of the DNA encoding bGLYAT ORF into pColdIII in the correct orientation and reading frame. bGLYAT was co-expressed with TF alone, or TF together with GroEL-GroES. In each case, the bGLYAT was insoluble according to SDS-PAGE results. Although bGLYAT could not be purified from other proteins, because it did not contain a tag, or seen to be soluble on SDSPAGE, a GLYAT enzyme activity test was performed on various fractions of the bacterially expressed protein lysates. Both soluble fractions of bGLYAT co-expressed with TF alone or TF together with GroEL-GroES had GLYAT enzyme activity. This is the first report of an enzymatically active recombinant bGLYAT. The effect of pH on the solubility of bGLYAT was briefly investigated but the results were not conclusive. In future studies, the bGLYAT should be expressed as a protein with a His or GST tag for purification and Western blotting performed on bacterial lysate fractions. The recombinant bGLYAT can also be used for future site-directed mutagenesis studies, thereby analysing the impact of SNPs on enzyme activity and amino acid specificity.