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Download (11Mb) A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/108813/ Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications STUDIES ON THE TARGETING AND PROCESSING OF PRORICIN. Michael Westby BSc. (Hons) Dunelm A thesis submitted for the degree of Doctor of Philosophy Department of Biological Sciences University of Warwick Coventry. U.K. September, 1991 CONTENTS Page No. Contents i-vii List of Figures viii-xi List of Tables xi Acknowledgements xii Declaration xiii Abbreviations xiv-xvi Dedication xvii Summary xviii î.o ihtbodpctioh 1 1.1 Summary 2 1.2 Plant storage proteins 3 1.2.1 Introduction 3 1.2.2 Classification of plant storage proteins 4 1.2.3 Storage proteins of cereals 4 1.2.4 Storage proteins of dicots. 4 1.2.5 Subcellular site of storage protein deposition- the protein bodies 9 1.2.6 Synthesis as preproproteins 11 1.2.7 Summary 15 1.3 Intracellular trafficking of newly-synthesised proteins 16 1.3.1 Introduction 16 1.3.2 Membrane translocation as a first step in compartmentalisation 16 1.3.3 Translocation across the ER membrane 18 1.3.4 Co-translatlonal modifications 21 1.3.5 Protein folding in the ER 21 1.3.6 Protein export from the ER 22 I 1.3.7 Control of ER export 24 1.3.8 Vacuolar targeting 26 1.3.9 Summary 30 1.4 Ricin and the castor bean 31 1.4.1 Introduction 31 1.4.2 HS-globulins 31 1.4.3 2S-albumins 32 1.4.4 7S-lectins - Ricin 32 1.4.5 Ricin - a typell RIP 36 1.4.6 Ricin and immunotoxins 38 1.4.7 Summary 39 1.5 Aims of the project 41 2.0 METHODS 2.1 Methods of DNA and RNA manipulation 43 2.1.1 Growth and Maintenance of bacterial cultures 43 2.1.2 Preparation of plasmid DNA 45 2.1.3 Storage of DNA 47 2.1.4 Qualitative estimation of DNA 47 2.1.5 Resolution of DNA using gel electrophoresis 49 2.1.6 Electrophoresis of RNA 52 2.1.7 Gel isolation of DNA fragments 52 2.1.8 Partitioning and precipitation of DNA 53 2.1.9 Restriction and modification of DNA 53 2.1.10 Transformation of E.coli 54 2.1.11 Sequencing of DNA 55 2.1.12 Site-directed mutagenesis 58 2.2 In vitro expression of preproricin clones 63 2.2.1 SP6/T7 in vitro transcription 63 2.2.2 Wheat germ lysate translations 64 2.2.3 Rabbit reticulocyte lysate translations 66 2.2.4 Xenopus egg cell-free translations 66 2.3 Analytical protein techniques 68 2.3.1 SDS-PAGE 68 2.3.2 Silver staining of SDS-polyacrylamide gels 68 2.3.3 Coomassie Blue staining of SDS-polyacrylamide gels 68 2.3.4 Western blotting of SDS-PAGE resolved proteins 68 2.3.5 Storage of SDS-polyacrylamide gels 70 2.3.6 Fluorography and autoradiography 70 2.3.7 TCA precipitation of protein samples 70 2.4 Transient expression in tobacco plant protoplasts 71 2.4.1 Introduction 71 2.4.2 Growth and maintenance of plants 71 2.4.3 Preparation of DNA 71 2.4.4 Isolation of protoplasts 72 2.4.5 Estimation of viable protoplasts 73 2.4.6 Protoplast transformation 73 2.4.7 Isolation and preparation of protoplast and media fractions 74 2.4.8 Enzymatic assaying of protoplast and media fractions 75 2.5 RNA N-Glycosidase activity of proricin 78 2.5.1 Preparation of aniline reagent 78 2.5.2 Incubation and extraction of ribosomes 78 2.5.3 Aniline treatment of modified rRNA 79 2.5.4 50Z Formamide/1.2% agarose gels 79 2.6 Isolation of protein body endoprotease activity and its assay 81 2.6.1 Growth of Ricinus communis plants 81 2.6.2 Preparation of extracts containing proricin- processing activity from developing seeds 81 2.6.3 Preparation of extracts containing proricin- processing activity from the protein bodies of dry seeds 81 • • • III 2.6.4 Removal of lectins from seed extracts using affinity chromatography on Sepharose-6B 82 2.6.5 Ammonium sulphate precipitation of seed extracts 82 2.6.6 Preparation of radiolabelled proricin from developing seed tissue 83 2.6.7 Synthesis of radiolabelled proricin from cDNA 84 2.6.8 Assay for proricin-processing activity 84 2.7 Expression of proricin in E.coli 85 2.7.1 Introduction 85 2.7.2 Maintenance and expression of JA221spINIIIompAproricin cultures 85 2.7.3 Preparation and analysis of fractions prepared from expression cultures 85 2.7.4 Beta-lactamase assay 86 2.8 Factor Xa and thrombin assays 87 3.0 CONSTRUCTION OF PREPR0RICIH/NPT2 CHIMERIC GENES IN A VECTOR SUITABLE FOR THEIR EXPRESSION IN PLANTS 88 3.1 Introduction 89 3.2 Aims 91 3.3 Approach 92 3.4 Computer analysis of the N-terminal region of preproricin 92 3.5 Cloning strategies 93 3.5.1 Source and characterisation of vectors 93 3.5.2 Construction of pCaMVNEOdeltaX 96 3.5.3 Construction of pPARTIAL 103 3.5.4 Construction of pFULL 103 3.6 Computer analysis of chimeric genes from pPARTIAL and pFULL 108 3.7 Tn vitro expression of chimeric gene from pPARTIAL 112 3.8 Discussion 114 ¡V 4.0 TRANSIENT EXPRESSION OF pPARTIAL AMD pFULL IN TOBACCO LEAF PROTOPLASTS 117 4.1 Introduction 118 4.2 Choice of marker enzyme and expression system 118 4.3 Isolation and transformation of protoplasts 120 4.3.1 Isolation of protoplasts 120 4.3.2 PEG/calcium nitrate-mediated transformation 123 4.4 Transformation of protoplasts with pCaMVNEO, pPARTIAL and pFULL 123 4.5 Protocol modifications and further transient expression studies 127 4.5.1 Amended approaches 127 4.5.2 Results from GUS assays 129 4.5.3 Results from npt2 assays 136 4.6 Discussion 142 5.0 BIOLOGICAL ACTIVITIES OF RECOMBINANT PRORICIN 147 5.1 Introduction 148 5.2 Aims and approach 149 5.3 Results 151 5.3.1 Source of recombinant proricin 151 5.3.2 Aniline assay 151 5.3.3 Results from the first experiment 153 5.3.4 Results from the second experiment 155 5.4 Discussion 158 6.0 IH VITRO PROCESSING OF RECOMBINANT PRORICIN USING A SOLOBLE MATRIX FRACTION FROM DRY CASTOR BEAM SEEDS 160 6.1 Introduction 161 6.2 Results 162 6.2.1 Source of proricin substrate 162 6.2.2 Transcription of proricin gene 164 6.2.3 Optimisation of wheat germ lysate 164 V 6.2.4 Protein body matrix fractionation 168 6.2.5 In vitro assay for processing activity 170 6.2.6 Removal of lectins by affinity chromatography 172 6.2.7 Effects of amino acid analogue incorporation /DDT on the processing of proricin ¿n vitro 174 6.2.8 Effects of EDTA and PMSF on enzyme activity. 176 6.2.9 In vitro processing of pulse-chased and immunoprecipitated proricin isolated from developing seeds 178 6.3 Discussion 180 7 . 0 CONSTROCTION OF PRORICIN CLOWES WITH LINKERS ENCODING FACTOR XA AND THBOMBIH RECOGNITION SEQUENCES 186 7.1 Introduction 187 7.2 Cloning and mutagenesis 190 7.2.1 Source of pINIIIompA2proricin 190 7.2.2 Cloning strategy 190 7.3 Expression in E.coli 196 7.4 Discussion 208 8 . 0 EXPRESSIOH OF PRORICIH AND PRORICIH MUTANTS IN VITRO USING A XENOPUS EGG CELL-FREE TRANSLATION SYSTEM 212 8.1 Introduction 213 8.2 Results 214 8.2.1 Cloning of Xa and Th mutant linkers into pGEMppRCL617 214 8.2.2 In vitro expression of preproricin and preproricin mutants 214 8.2.3 Proteolytic cleavage of proricin and proricin mutants synthesised in a wheat germ lysate 218 8.2.4 Cloning of preproricin and preproricin mutant clones into pSP64T 221 8.2.5 Expression of preproricin and preproricin mutants in Xenopus egg cell-free translation system 222 vj 8.2.6 Glycosylation and lectin activités of recombinant proricin and proricin mutants 225 8.2.7 Specific proteolytic cleavage of recombinant proricin Xa mutant 229 8.3 Discussion 229 9.0 GENERAL DISCUSSION 235 APPENDICES: 240 Appendix A: cDNA sequence and deduced protein sequence of preproricin Appendix B: Restriction map of ppRCL617 Appendix C: Vectors Appendix D: Computer programme for finding likely sites of signal peptide cleavage Appendix E: Suppliers BIBLIOGRAPHY 247 VII FIGURE LEGENDS SCHEMATIC REPRESENTATION OF THE 3D STRUCTURE OF RICIN AND RCA. 34 SITE OF ACTION OF RICIN ON RAT 28S rRNA. 37 VON HEIJNE PLOT OF N-TERMINAL REGION OF PREPRORICIN 94 RESTRICTION MAP AND PARTIAL DNA SEQUENCE OF pCaMVNEO 97 CLONING STRATEGY FOR pCaMVNEOAX. 98 HYBRIDISATION SCREENING OF PUTATIVE XHOI MUTANTS USING THE 32P-LABELLED MUTAGENIC OLIGONUCLEOTIDE, 0AB1049. 101 DNA SEQUENCE ANALYSIS OF PUTATIVE XHOI MUTANT CLONES B8, BIO, C3, C9 AND D7. 102 RESTRICTION DIGEST ANALYSIS OF pCaMVNEOAX SHOWING THE CORRECT ORIENTATION OF THE 1KB BAMHI FRAGMENT (CONTAINING THE MUTATED NPT2 GENE) 104 CLONING STRATEGY FOR pPARTIAL CONSTRUCT. 105 CLONING STRATEGY FOR pFULL CONSTRUCT. 106 HYBRIDISATION SCREENING OF PUTATIVE SALI MUTANTS USING THE 32P-LABELLED MUTAGENIC OLIGONUCLEOTIDE, OAB1179.
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