Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. The Refolding of Recombinant Human Liver Methylmalonyl-CoA Mutase from Inclusion Bodies Produced in Escherichia coli A thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in Biochemistry at Massey University Michelle Marie Haves 1998 n ABSTRACT Human methylmalonyl-CoA mutase (hMCM) is an adenosylcobalamin-dependent enzyme that catalyses the structural rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA as part of the catabolism of the branched chain amino acids valine, leucine and isoleucine, odd chain fatty acids and intermediates of cholesterol metabolism. Reactions that require adenosylcobalamin (AdoCbl) have been intensively studied, and the first step in the catalysis is widely agreed to involve homolytic cleavage of the unusual carbon-cobalt bond in the cofactor. A reliable source of recombinant hMCM would be useful in defining more fully the mechanistic pathway of AdoCbl-dependent enzymes. Recombinant hMCM overexpressed in E. coli forms insoluble aggregates of inactive protein known as inclusion bodies. hMCM inclusion bodies were purified, solubilised and then several different in vitro refolding techniques were tested in attempts to produce active recombinant hMCM from purified solubilised inclusion body material. These methods included refolding by rapid dilution, refolding by dialysis, detergent-assisted refolding, refolding by gel filtration chromatography and chaperonin-assisted refolding. Chaperonin-assisted refolding necessitated the strain DHl/pGroESL. Refolding by rapid dilution of the GdmHCl-solubilised inclusion bodies into a refolding buffer was judged to be the simplest and most effective method, however the refolding process was extremely inefficient. Refolding by rapid dilution was scaled up to 2 litres to produce as much active hMCM as possible. The refolded protein was concentrated by batch adsorption to and stepwise elution from hydroxyapatite, and further purified using a synthesised 5' adenosylcobalamin­ agarose 'affinity' chromatography column. The final refolded hMCM preparation contained a single ~ 29 kDa contaminant protein, tentatively identified as E. coli branched-chain amino acid aminotransferase (EC 2.6.1.42), present in approximately equal amounts to the hMCM, and had a specific activity of ~ 3.11 units/mg. iii ACKNOWLEDGEMENTS Firstly I would like to thank my supervisor Mark Patchett, who was continually there with wonderful ideas and practical assistance in the lab, for his advice during the research project and also during the writing of this thesis. I would like to acknowledge Jo Mudford for N-terminal sequencing, and Carole Flyger for running the Twilight Zone lab so smoothly. Thanks also to John Tweedie for computing advice. Thanks to my friends and colleagues in the Twilight Z.One, and others around the Biochemisty Department, and University who have made the last few years so enjoyable. Also thankyou to all my flatmates, who I have learnt so much from Thanks to my family. Mum, Dad, Mark, Claire, Paul, Adrian and Ann for their constant suppon throughout my time at Massey. Extra special thanks must go to Terry and Geordi for their encouragement and understanding while I was writing, and for believing me whenever I said it was ''just about .finished'! Thank God for methylmalonyl-CoA mutase; it is a stubborn but special little protein. And finally thanks to anyone who reads this line and has picked up this thesis hoping that they will find the information they need. Good luck! LIST OF ABBREVIATIONS S'AdoCbl Amp Avg bMCM BSA C-terminal Cs-(3-D Gluc CAM CAPS cDNA CHAPS CHAPSO CMC CNCbl CTAB EDTA ER g GdmHCI hMCM HTP kan KP LDAO LM LubrolPX MAP 5' deoxyadenosylcobalamin Ampicillin Average Bacterial methylmalonyl-CoA mutase Bovine serum albumin Carboxyl terminal Cs-(3-D Glucopyranoside Chloramphenicol 3-[ Cyclohexylaminol }-1-propanesulfonic acid Complementary DNA 3-[ (3-Cholamidopropyl)dimethylarnmonio ]-1- propanesulfonate 3-[(3-Cholamidopropyl)dimethylammonio]-2- hydroxypropane-1-sulfonate Critical micelle concentration l.. 'yanocobalamm Cetyltrimethylammonium bromide Ethylenediaminetetraacetic acid Endoplasmic reticulum Gravitational field, unit of Guanidinium hydrochloride Human methylmalonyl-CoA mutase H ydroxyapatite Kanamycin Potassium phosphate Lauryldimethylamine oxide Lauryl Maltoside Polyoxyethylene (9) lauryl ether Methionine aminopeptidase 1V MEGA-9 MCM Mr mt NADH Nonidet P40 N-terminal PAGE PDI PK PMSF PPI RNase SDS TCA cycle TEMED Tris Triton X-lUU Tween 20 Z-3-XX Nonanoyl-N-methylglucamide Methylmalonyl-CoA mutase Relative molecular mass Mitochondrial Nicotinamide-adenine dinucleotide, reduced Nonaethylene glycol octylphenyl ether Amino terminal Polyacrylamide gel electrophoresis Protein disulphide isomerase Protein Kinase Phenylmethylsulphonyl fluoride Peptidyl-prolyl cis-trans isomerase Ribonuclease Sodium dodecyl sulphate Tricarboxylic acid cycle N, N, N', N'-tetramethylethylenediamine Tris-(h ydroxymethyl)-aminomethane Nonaetnylene g1yco1 octy1pneny1 ether Polyoxyethylene sorbitan monolaurate Zwittergent 3-:XX series V V1 THREE AND ONE LETTER AMINO ACID CODE Ala A Alanine Arg R Arginine Asn N Asparagine Asp D Aspartic acid Cys C Cysteine Gin Q Glutamine Glu E Glutarnic acid Gly G Glycine His H Histidine Ile I Isoleucine Leu L Leucine Lys K Lysine Met M Methionine Phe F Pheny !alanine Pro p Proline Ser s Senne Thr T Threonine Tyr y Tyrosine Val V Valine Trp w Tryptophan Vil TABLE OF CONTENTS Title Page i Abstract ii Acknowledgements Ill List of Abbreviations lV Three and One Letter Amino Acid Code vi Table of Contents vii List of Figures Xlll List of Tables xvii CHAPTER ONE INTRODUCTION 1 1.1 Methylmalonyl-CoA mutase 1 1.2 The Formation of Inclusion Bodies 4 1.:.5 t'rotem Keto1e1mg .t rom 1ncms1on Hoay Matenat '.J 1.3.1 Differences Between Refolding In Vitro and Folding In Vivo 9 1.3.2 Refolding by Rapid Dilution 10 1.3.3 Refolding by Dialysis 14 1.3.4 Detergent-Assisted Refolding 16 1.3.5 Refolding by Size-Exclusion Chromatography 19 1.3.6 In Vitro Chaperonin-Assisted Refolding 20 1.3.7 Co-expression of Molecular Chaperonins In Vivo 24 1.4 Aims of This Project 25 CHAPTER TWO MATERIALS AND METHODS 27 2.1 Co-expression of Chaperonins In Vivo with hMCM 27 2.2 Expression of hMCM Inclusion Bodies for Refolding 28 2.2.1 Comparison of Two hMCM Expression Systems 2.2.2 Large-Scale Growth of SRP84/pGPl-2/pMEXHCO and Induction of Recombinant hMCM Expression 2.3 Preparation of hMCM Inclusion Bodies for Refolding Experiments 2. 3 .1 Isolation of Inclusion Bodies by Differential Centrifugation 2.3.2 Solubilisation of Inclusion Bodies iniciai 111viC1vi nxauiw.ui; i:A.,~l im~lll..'., 2.4.1 Initial Small-Scale Rapid Dilution Experiments 2.4.2 Scale-up of Small-Scale Rapid Dilution Experiments 2.4.3 Dialysis of Solubilised Protein 2.4.4 Detergent-Assisted Refolding by Rapid Dilution 2.4.5 Time Trial Refolding Experiment Over 24 Hours 2.4.6 Dimerization Experiment 2.4. 7 Refolding by Gel Filtration Chromatography 28 29 30 30 31 32 32 33 33 34 34 36 Vl11 IX 2.5 Concentration of Refolded Protein with Hydroxyapatite 37 2.5.1 Hydration of Hydroxyapatite 37 2.5.2 Adsorption of Protein to Hydroxyapatite 37 2.5.3 Elution of Protein from Hydroxyapatite 38 2.6 Synthesis and Testing of 5' AdoCbl-agarose for Affinity Chromatography 38 2.6.1 Synthesis of 5' AdoCbl-agarose Resin 38 2.6.2 Scanning of Prepared 5' AdoCbl-agarose Resin 39 2.6.3 Testing Reversible Adsorption of MCM to the Prepared 5' AdoCbl-agarose Resin 39 2.6.4 Conditions for Effective hMCM Adsorption to 5' AdoCbl-agarose 41 2.7 Large-Scale Refolding and Purification of hMCM 41 2.7.1 Inclusion Body Purification, Solubilisation and Refolding 41 L/.L tlatcn Aasorpnon to ana ::>tepwise r.muon from hydroxyapatite 42 2.7.3 5' AdoCbl-agarose ' Affinity ' Chromatography 42 2.7.4 Concentration of the Pooled 'Affinity' Chromatography Fraction 43 2.8 Purification and Use of Recombinant GroEL and GroES 44 2.8.1 Ammonium Sulfate Precipitation Method 44 2.8.2 Cell Lysis, Clarification of Extract and First DEAE-Sephacel Step 44 2.8.3 Continued Purification of GroES 45 2.8.4 Continued Purification of GroEL 46 X 2.8.5 Chaperonin-Assisted Refolding of ~-galactosidase 47 2.8.6 Chaperonin-Assisted Refolding of hMCM 49 2.9 Protein Electrophoresis, Electroblotting and N-terminal Sequencing 49 2.9.1 Treatment of Samples Taken from Cultures for SDS-PAGE 49 2.9.2 Discontinuous SDS-Polyacrylamide Gel Electrophoresis 50 2.9.3 Drying of SDS-Polyacrylamide Gels 52 2.9.4 Electroblotting for N-terminal Sequence Analysis 52 2.9.5 N-terminal Sequencing 54 2.10 Protein Determination and Activity Assay Methods 54 2.10.1 Determination of Protein Concentration 54 2.10.2 Succinyl-CoA Preparation for use in hMCM Activity Assay 54 2.10.3 Enzyme Coupled Assay for Methylmalonyl-CoA mutase Activity 56 2.10.4 ~-galactosidase Activity Assay 57 CHAPTER THREE RESULTS 3.1 Purification of hMCM Inclusion Bodies 59 3.2 Comparison of Two hMCM Expression Systems 61 3.3 Large-Scale Growth and Induction of SRP84/pGP1-2/pMEXHCO and Induction of Recombinant hMCM Expression 61 3.4 Large-Scale hMCM Inclusion Body Purification 64 3.5 Determination of the Best of Three Different Solubilisation Solutions 3.6 The Effect of Protein Concentration on Refolding by Rapid Dilution 3.7 Scale up of Rapid Dilution Experiments 3.8 Time Trial Experiment 3.9 Dimerization Experiment 3.10 Detergent-Assisted Refolding 3.11 Protein Refolding by Dialysis Against a Refolding Buffer 3.12 Refolding by Gel Filtration Chromatography 3.13 E.coli GroEL and GroES Chaperonin Purification 3.14 Testing Purified GroEL and GroES for Biological Activity 3.15 Chaperonin-Assisted Refolding of Solubilised hMCM Inclusion Body Material 3.16 Co-expression of Chaperones In Vivo with hMCM 3.17 Synthesis of 5' deoxyadenosylcobalamin-agarose for Affinity Chromatography 3.18 UV-Visible Spectroscopy of Control Cobalamin Solutions and Prepared 5' AdoCbl-agarose Resin 3.19 Solubilisation and Refolding of hMCM Inclusion Bodies on a Large-Scale 3.20 hMCM Purification by Affinity Chromatography 3.21 Further Purification and Concentration of the Two hMCMPools CHAPTER FOUR DISCUSSION 4.1 Preparation of hMCM Inclusion Bodies 4.2 Refolding of hMCM by Rapid Dilution 64 67 67 69 71 75 79 79 80 96 98 101 102 105 108 114 114 119 119 119 Xl xii 4.3 hMCM Refolding by Dialysis Against a Refolding Solution 120 4.4 Detergent-Assisted Refolding of hMCM 120 4.5 Refolding by Gel Filtration Chromatography 121 4.6 Dimerization Experiment 122 4.7 Chaperonin-Assisted Refolding 123 4.7.1 In Vitro Chaperonin-Assisted Refolding 123 4.7.2 In Vivo Chaperonin-Assisted Refolding 125 4.8 Production of Recombinant hMCM From Inclusion Bodies in E.coli 126 4.9 Affinity Chromatography 128 4.10 Future Work 129 APPENDIX I 131 Plasmids for hMCM Expression in SRP84/pGPl-2/pMEXHCO 131 APPENDIX2 132 Edited SWISS-PROT Database Entry of hMCM 132 REFERENCES 135 xiii LIST OF FIGURES FIGURE PAGE 1.1 A schematic view of the structure of the active a chain of 2 methylmalonyl-CoA mutase from Propionibacterium shennanii 1.2 The reaction pathway involved in the interconversion of 3 succinyl-CoA and propionyl-CoA 1.3 hMCM-catalysed reaction 5 1.4 Possible fates of an over-expressed protein in E.coli 1.5 Flow chart showing the possible outcomes in vitro refolding 11 1.6 lliustration of the effect of protein concentration on the 12 refolding of rhodanese 1.7 lliustration of the temperature dependence of renaturation 13 1.8 lliustration of the concentration dependence of functional 13 renaturation 1.9 The effect of the addition of L-arginine to the refolding 14 solutions, on the yield of renaturation 1.10 Percent reactivation vs rhodanese concentration after 15 denaturation in guanidine and subsequent dialysis 1.11 Schematic diagram of detergent monomers and micelles in 17 solution 1.12 Schematic representation of protein refolding within size- 20 exclusion chromatography media 1.13 Effect of order of addition of GroES and substrate on 22 protection of substrate from proteolysis XIV 3.1 SDS-PAGE analysis of samples taken during the small-scale 60 preparation of inclusion bodies 3.2 SDS-PAGE analysis of samples taken after induction of two 62 hMCM E. coli expression systems 3.3 SDS-PAGE analysis of samples taken during large-scale 63 growth and induction of E.coli SRP84/pGPl-2/p~XHCO 3.4 SDS-PAGE analysis of large-scale inclusion body purification 65 3.5 Graph showing the hMCM activity recovered after rapid 68 dilution experiments over a range of final protein concentrations 3.6 Plot of the gain in hMCM activity during 24 hours on ice 70 3.7 Graph of hMCM specific activity in fraction 12 vs the number 72 of hours since fraction 12 was eluted from hydroxyapatite 3.8 A2so nm profile of fractions collected during hMCM refolding 78 by gel filtration chromatography 3.9 SDS-PAGE analysis of fractions collected during refolding by 81 gel filtration chromatography 3.10 SDS-PAGE analysis of samples collected during purification 83 of GroES and GroEL on a DEAE-Sephacel column 3.11 SDS-PAGE analysis of fractions 81 to 97 collected during 84 purification of GroES and GroEL on a DEAE-Sephacel column 3.12 SDS-PAGE analysis of fractions 66 to 78 and 100 to 112 85 collected during purification of GroES and GroEL on a DEAE-Sephacel column 3.13 SDS-PAGE analysis of the E.coli chaperonin GroES (Amrein 86 et al., 1995) xv 3.14 SDS-PAGE analysis of fractions 48, 50, 52, 54, 56, 58, 60, 62 87 and 64 collected during the purification of GroES and GroEL on a DEAE-Sephacel column 3.15 SDS-PAGE analysis of fractions collected during the second 88 DEAE-Sephacel chromatography step in the GroES purification 3.16 SDS-PAGE analysis of more fractions collected during the 89 second DEAE-Sephacel chromatography step in the GroES purification 3.17 Further SDS-PAGE analysis of fractions collected during the 90 second DEAE-Sephacel chromatography step in the GroES purification 3.18 SDS-PAGE analysis of fractions collected during elution of 92 the HiLoad 26/10 Q-Sepharose high performance column, during the GroES purification 3.19 SDS-PAGE analysis of fractions collected during Sephacryl 93 S300 chromatography of the first half of the GroEL sample 3.20 SDS-PAGE analysis of fractions collected during the 94 Sephacryl S300 chromatography of the second half of the GroEL sample 3.21 Combined GroEL and GroES purification; SDS-PAGE 95 analysis 3.22 Absorbance spectra of a 0.5 mM control solution of 5' AdoCbl 106 3.23 Absorbance spectra of the prepared 5'AdoCbl-agarose resin 107 3.24 SOS-PAGE analysis of fractions collected during stepwise 110 elution from hydroxyapatite 3.25 3.26 3.27 SDS-PAGE analysis of fractions collected during stepwise salt elution of the 5'AdoCbl-agarose affinity chromatography resin SDS-PAGE analysis of the concentrated hMCM pools and the concentrated low salt pool filtrate SDS-PAGE analysis of samples taken throughout the large­ scale hMCM inclusion body purification, solubilisation, refolding and further purification XVl 113 115 117 LIST OF TABLES FIGURE 2.1 3.1 3.2 3.3 3.4 3.5 3.6a 3.6b 3.7 3.8 3.9 3.10 Detergent concentrations used in refolding solutions Refolding by rapid dilution of hMCM inclusion bodies solubilised with three different solubilising agents 100-fold rapid dilutions of hMCM inclusion bodies, solubilised at two different final GdmHCl concentrations, and assayed in duplicate for hMCM activity Effect of protein concentration on activity regain when refolding by rapid dilution into a non-denaturing buffer or refolding solution hMCM activity assays of fractions eluted from hydroxyapatite hMCM activity assays of HTP fraction 12 over 189 hours hMCM refolding by rapid dilution using detergent- supplemented refolding buffers hMCM refolding by rapid dilution using detergent- supplemented refolding buffers Refolding by dialysis at three different protein concentrations Refolding of E.coli ~-galactosidase in the presence of GroES and GroEL Chaperonin-assisted refolding with incomplete basic refolding solutions Chaperonin-assisted hMCM refolding by rapid dilution XV1l PAGE 35 66 67 69 73 74 76 77 79 97 99 100 XVlll 3.11 Batch adsorption results 103 3.12 Elution of hMCM from 5'AdoCbl-agarose 104 3.13 Different elution conditions trialled to elute hMCM from the 105 prepared 5' AdoCbl-agarose 3.14 Colour of neutral and acidified cobalamin control solutions, 109 prepared 5'AdoCbl-agarose and cyanocobalamin-agarose 3.15 Fractions collected during elution of protein from 101 hydroxyapatite 3.16 Fractions collected during purification by affinity 112 chromatography of large-scale refolded hMCM