Molecular characterisation of glycine-N-acyltransferase from two primates: the vervet monkey and the chacma baboon by Cornelius Mthiuzimele Mahlanza Hons. B.Sc (Biochemistry) Dissertation submitted in partial fulfilment of the requirements for an Masters degree in Biochemistry Division for Biochemistry, School of Physical and Chemical Sciences, North-West University, Potchefstroom Campus, Potchefstroom, 2520, South Africa Supervisor: Prof. A.A. van Dijk March 2011 1 Acknowledgements I would like to thank God for the strength he gave me throughout this project. I would like to thank my study leader (Prof Albie van Dijk) for her efforts in supervising this project and Mr Jaco Wentzel for helping with the translation of the abstract from English to Afrikaans. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. I thank NRF (FA grant: FA200503170001), DST-NRF BioPAD project (BPP007) and the North-West University Centre for Human Metabonomics (NWU-CHM) for their financial assistance. I hereby appreciate all the help that I received from my fellow postgraduate students in the Biochemistry Department (North-West University). 2 This project started as a dream coming true. Then, it shifted from being my MSc project to being my introduction to strangers, my personality and ambition then finaly it became my life. Now, it is over. I have to dream again so that I can start a new life for myself. 3 Table of contents Abstract .......................................................................................................................... 9 Opsomming ................................................................................................................. 12 Chapter 1 ...................................................................................................................... 15 Introduction and literature review.............................................................................. 15 1.1 Introduction ........................................................................................................... 15 1.2 Metabolism and inborn errors of metabolism..................................................... 16 1.3 Detoxification enzyme systems ........................................................................... 18 1.3.1 Description of detoxification .............................................................................. 18 1.3.2 Detoxification enzymes ..................................................................................... 18 1.4 Single nucleotide polymorphism ......................................................................... 20 1.5 Protein classification and the GNAT superfamily .............................................. 22 1.6.1 Reaction localisation and description ................................................................ 24 1.6 Glycine-N-acyltransferase .................................................................................... 24 1.6.2 GLYAT substrates ............................................................................................ 25 1.6.3 The role of GLYAT and glycine in non-detoxification pathways ........................ 27 1.6.4 GLYAT family members .................................................................................... 28 1.7 Recombinant protein expression ......................................................................... 29 1.7.1 The expression system host ............................................................................. 29 1.7.2 The pCold protein expression system and molecular chaperones.................... 30 1.7.3 Protein purification ............................................................................................ 30 1.7.3.1 Ammonium sulphate precipitation .............................................................. 31 1.7.3.2 Nickel affinity histidine tag purification ........................................................ 32 1.8 Problem formulation and aims ............................................................................. 33 Chapter 2 ...................................................................................................................... 35 4 DNA sequencing and analysis of the open reading frames that encode GLYAT of the chacma baboon and the vervet monkey ............................................................. 35 2.1 Introduction ........................................................................................................... 35 2.2 Materials and methods ......................................................................................... 38 2.2.1 Cloning of cDNA encoding chacma baboon GLYAT ......................................... 38 2.2.1.1 Ethical approval .......................................................................................... 38 2.2.1.2 Extraction of total RNA from chacma baboon liver ..................................... 38 2.2.1.3 Determination of the concentration and purity of nucleic acid samples ...... 39 2.2.1.4 Characterisation of nucleic acids on agarose gel electrophoresis .............. 40 2.2.1.4.1 Agarose gel electrophoresis for DNA separation ................................. 40 2.2.1.4.2 Agarose formaldehyde denaturing gel electrophoresis for RNA separation ............................................................................................ 41 2.2.1.5 cDNA synthesis .......................................................................................... 42 2.2.1.6 Polymerase chain reaction ......................................................................... 44 2.2.1.7 PCR clean up and gel extraction ................................................................ 46 2.2.1.8 Cloning of a PCR product containing an ORF encoding for GLYAT of chacma baboon ...................................................................................... 47 2.2.1.8.1 TA cloning of the PCR product containing an ORF encoding for GLYAT of chacma baboon ............................................................................... 47 2.2.1.8.2 Directional cloning of PCR amplicons containing the ORF that encodes for GLYAT of chacma baboon into pColdIII vector .............................. 50 2.2.1.8.3 Preparation of chemically competent Esherichia coli cells ................... 52 2.2.1.8.4 Transformation .................................................................................... 53 2.2.1.8.5 Selection of successfully transformed bacteria .................................... 55 2.2.1.8.6 Extraction of plasmids from transformed cells using the PureYield™ Plasmid Midi preparation system ......................................................... 57 2.2.1.8.7 Restriction enzyme digestion reaction ................................................. 58 2.2.1.8.8 Ligation reaction .................................................................................. 59 5 2.2.2 Reconstruction of the vervet monkey GLYAT open reading frame ................... 59 2.2.2.1 Preparation of leukocytes from whole blood of vervet monkey .................. 59 2.2.2.2 Genomic DNA extraction from leukocytes of a vervet monkey................... 60 2.2.3 DNA sequencing ............................................................................................... 60 2.3 Results ................................................................................................................... 62 2.3.1 Extraction of total cellular RNA from chacma baboon liver ............................... 62 2.3.2 cDNA synthesis using oligo (dT) primers .......................................................... 63 2.3.3 Amplification and gel extraction of an amplicon containing the open reading frame encoding GLYAT of the chacma baboon ................................................ 64 2.3.4 Cloning of a PCR amplicon encoding chacma baboon GLYAT open reading frame into a TA vector ....................................................................................... 67 2.3.5 Transformation of pBabTAV001, pColdIII and pColdIII-EHDV into JM109 cells followed by plasmid midi preparation ................................................................ 68 2.3.6 Restriction enzyme digestion of pBabTAV001, pColdIII and pColdIII-EHDV .... 70 2.4. Amplification of a fragment across the position where human primers were bound on the chacma baboon GLYAT cDNA to confirm human primer sequence by DNA sequencing ............................................................................ 77 2.5 The extraction of genomic DNA from leucocytes of vervet monkey ................ 79 2.6 DNA sequencing .................................................................................................... 81 2.7 Summary ................................................................................................................ 94 Chapter 3 ...................................................................................................................... 97 Expression of a chacma baboon’s recombinant glycine-N-acyltransferase in Escherichia coli and the preparation of a crude cytoplasmic extract containing native glycine-N-acyltransferase from chacma baboon liver .................................. 97 3.1 Introduction ........................................................................................................... 97 3.2 Material and methods ........................................................................................