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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type ofcomputer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back ofthe book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. MI 48106-1346 USA 313f761-4700 800:521-0600 MOLECULAR CLONING AND EXPRESSION OF THE SEA URCHIN DYNEIN BETA-HEAVY CHAIN A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOMEDICAL SCIENCES (BIOCHEMISTRY) DECEMBER 1995 By Hening Ren Dissertation Committee: Ian R. Gibbons, Chairperson Frederick C. Greenwood Marguerite Volini David Jameson Heinz Gert de Couet UMI Number: 9615548 UMI Microform 9615548 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ACKNOWLEDGEMENTS I would like to give my sincere thanks to many people who have made my graduate tenure enjoyable. My first thanks goes to the secretaries in the Department of Biochemistry and Biophysics and Ke\'lalo lab: June Ko, Frances Okimoto and Geri Mitomi who, with their professionalism, have navigated me through the sea of paperwork and a lot of other helps. I would like to thank Rich Chock and Steve Sakata for the work they did to ensure the smooth running of experiments all year round. To the members in the Gibbons lab, I would like to give my deepest gratitude for their tutelage, friendship and help, in particular: Dr. Grace Tang for her extensive knowledge on the biochemistry and laboratory techniques of dynein, and her advice; Dr. Barbara H. Gibbons for her intellectual insight, and teaching on several experiments; Cheryl Phillipson for her immense help on laboratory techniques and supplies; Dr. Garbor Mocz for many helpful discussions and analysis of CD data. My special thanks also goes to Dr. Grace Tang and Cheryl Phillipson for their critical reading of this dissertation. I am deeply in debt to Dr. Ian Gibbons who has guided me, encouraged me and financially supported my research in the past six years. He has also greatly improved my English writing. My sincere thanks goes to Drs. Margueate Volini, David Jamenson, Frederick Greenwood and H. Gert deCouet for teaching and serving on my committee. I should also thank Dr. Eric Rosenthal I who served in my committee until his departure to California, for much help and advice. iii Finally, I would like to thank my parents for their dedication towards my education; Hexuan Ren, my brother, for his support; and Qin Zhou, my wife, for her understanding and tremendous support. iv ABSTRACT The DNA sequence that encodes sea urchin dynein ~-heavy chain (~-HC) was cloned into a plasmid vector. Plasmid pDl. 3 contains a single 13,168 bp cDNA insert that encodes 98% of the ~-HC. The identity of the cDNA insert was verified by partial sequencing and restriction digestion, and was found in agreement with that predicted from the published sequence determined by a PCR based method. Polypeptides encoded by the cloned cDNA reacted positively with antibodies raised against native dynein. Truncated heavy chains of up to 300 kDa were expressed in E. coli, and some were purified under denaturing conditions and refolded. Refolding of one of them, designated RBs12 and representing amino acid residues 2,992-3,501 in the ~-HC, yielded a stable soluble protein with possible native-like conformation as judged by its circular dichroic spectrum and solubility in water. Connecting the four nucleotide binding sites in the middle of ~-HC with the C-terminal portion of the protein, this region contains two hypothetical coiled-coil structures that was proposed to form the short projection on the dynein head. No specific association of RBs12 with in vitro polymerized microtubules was observed, nor did it significantly block the restoration of beat frequency in KCl­ extracted sea urchin sperm flagella by native dynein. The nucleotide binding domains of dynein ~-HC were identified by analyzing the homology between dynein isoforms. v The putative ATP binding/hydrolysis domain and a second NTP binding domain were expressed in E. coli as fusion or non­ fusion proteins. Soluble thioredoxin-fusion proteins were obtained using a low level expression vector, accompanied by high level co-expression of E. coli chaperon 70 from another co-transformed plasmid. The soluble thioredoxin-fusion proteins, purified by Ni-chelating chromatography, were prone to precipitate upon exposure to reduced pH, heat or after prolonged storage. Photo-cleavage of fusion protein representing the ATP binding domain was observed in medium containing millimolar concentrations of vanadate upon irradiation at 365 nm, indicating the presence of native-like conformation. It did not significantly affect the motility of reactivated sea urchin sperm, but slightly inhibited the restoration of beat frequency of KCl extracted sperm by native d}~ein. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS iii ABSTRACT. v LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii CHAPTER I. INTRODUCTION 1 CHAPTER II. MATERIALS AND METHODS 16 A. Construction of gene-specific cDNA library and isolation of dynein cDNA clones 16 1. Isolation of poly A+ RNA 16 2. Construction of gene-specific cDNA library 18 3. Transformation of E. coli host 21 4. DNA probe synthesis 22 5. Screening of recombinants by colony hybridization 25 6. Plasmid preparations 26 7. DNA sequencing 28 B. Construction of expression clones and expression of recombinant proteins 30 1. Isolation of DNA fragments 30 2. Generation of expression construct 32 3. Transformation of E. coli host 33 4. Screening and characterization of expression clones 33 5. Large scale culture and expressing of fusion proteins 35 C. Purification of fusion protein 36 1. Lysis of bacterial cells 36 2. Purification of recombinant protein under denaturing conditions 37 3. Purification of recombinant protein under native conditions 38 vii D. studies of the properties of the soluble recombinant protein 38 1. Sucrose density gradient 38 2. Vanadate-mediated photo-cleavage 39 3. Effect on the motility of reactivated sperm 39 4. Microtubule co-sedimentation assay 40 5. Circular dicroic study 41 CHAPTER III. RESULTS. .. 42 A. Isolation and characterization of eDNA clones 42 1. Synthesis of eDNA and construction of eDNA library 42 2. Screening of the eDNA libraries and characterization of eDNA clones ........ 43 3. Assembly of a near full-length eDNA clone 46 B. Expression and purification of recombinant proteins: the large fragments 47 1. Expression of recombinant protein in pETS vectors 47 2. Expression of recombinant protein in pET24 vectors 49 3. Refolding of AN1480 and P748 .... 51 C. Expression and purification of NTP binding domains 52 1. Identification of the NTP binding domains 53 2. Expression of NTP binding domains in pET24 vector 54 3. Expression 0f the NTP binding domains as thioredoxin or glutathione-S-transferase fusion proteins 56 4. Expression of recombinant proteins in a low copy number plasmid vector . 58 D. Properties of the soluble recombinant proteins 60 1. The aggregation status of the soluble fusion protein 60 2. Vanadate mediated cleavage ..... 61 3. Effect on the motility of reactivated sea urchin sperm 62 4. Effect on in vitro polymerized microtubles 62 5. Circular Dichroism spectra . 63 viii CHAPTER IV. DISCUSSION . 110 A. Isolation and characterization of cDNA clones 110 B. Expression and purification of recombinant proteins 111 C. Properties of soluble recombinant proteins 120 CHAPTER V. CONCLUSIONS. 132 APPENDICES 134 Appendix I. Synthetic oligonucleotides used in this study 134 Appendix II. Alignment of nucleotide binnding domains in dynein heavy chains 135 BIBLIOGRAPHY 145 ix LIST OF TABLES Table Page 1­ Summary of cDNA libra~ies 64 2. Summary of cDNA clones 65 3. Summary of large dynein ~-HC expression constructs 84 x LIST OF FIGURES Figure Page Figure 1..1. The structure of an idealized axoneme 12 Figure 1..2 Three-headed outer arm dynein molecules in the bouquet configuration 13 Figure 1..3 Schematic diagram of a three-headed outer arm dynein interacting with two doublet microtubules of a flagellar axoneme .. 14 Figure 1..4 A linear map of sea urchin dynein ~-heavy chain 15 Figure 3.1 Autoradiograph of cDNAs separated on agarose gel 66 Figure 3.2 Chromatography of cDNA on Sephacryl H-500 column 67 Figure 3.3 EcoRI restriction digestion analysis of plasmids 68 Figure 3.4 ApaI/NotI restriction digestion analysis of plasmids ............. 70 Figure 3.5 Schematic diagram for the assembly of pD1.3 72 Figure 3.6 Restriction digestion analysis of pD1.3 74 Figure 3.7 Analysis of expression clone e12 75 Figure 3.8 Analysis of ER1470/BL21(DE3) low temperature induction 77 Figure 3.9 Analysis of pER1470/pGroESL/BL21 (DE3) induction 79 Figure 3.1.0 Western blot analysis of RB512 ..
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