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Research Collection Master Thesis Generation and characterization of monospecific antibodies against Sm- and LSm-Proteins Author(s): Hänni, Sandra Silvia Publication Date: 2002 Permanent Link: https://doi.org/10.3929/ethz-a-004432542 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Departement Angewandte Biowissenschaften Institut für Pharmazeutische Wissenschaften Generation and Characterization of monospecific antibodies against Sm- and LSm- Proteins Diploma Thesis of Sandra Silvia Hänni Citizen of Gurzelen BE Supervisor: Prof. Dr. P. A. Schubiger Supervisor: Dr. C. Kambach March 4 – July 26 2002 Diploma Thesis Structural Biology Sandra Haenni Paul Scherrer Institute TABLE OF CONTENTS 1. SUMMARY .....................................................................................................................................................4 2. INTRODUCTION ............................................................................................................................................5 2.1 Aim of the project .......................................................................................................................................5 2.2 RNA Splicing and Sm- Proteins ................................................................................................................5 3. MATERIALS AND METHODS.......................................................................................................................8 3.1 Construction of Expression vectors ........................................................................................................8 3.1.1 D2D1(78):pRK172...................................................................................................................................9 3.1.2 D2(27-118)D1:pRK172..........................................................................................................................11 3.2.3 D3B(91):pQE30 ....................................................................................................................................12 3.1.4 D3(75)B:pQE30 ....................................................................................................................................12 3.2 Protein Expression and Purification ......................................................................................................13 3.2.1 D2D1(78):pRK172.................................................................................................................................13 3.2.2 D2(27-118)D1:pRK172..........................................................................................................................14 3.3 Phage Library Screening .........................................................................................................................15 4. RESULTS.....................................................................................................................................................18 4.1 Cloning of Expression vectors ...............................................................................................................18 4.1.1. D2D1(78):pRK172................................................................................................................................18 4.1.2 D2(27-118)D1:pRK172..........................................................................................................................18 4.1.3 D3B(91):pQE30 ....................................................................................................................................19 4.1.4 D3(75)B:pQE30 ....................................................................................................................................20 4.2 Protein Expression and Purification ......................................................................................................20 4.2.1 Protein Expression of D2D1(78):pRK172 .............................................................................................20 4.2.2 Protein Expression of D2(27-118)D1:pRK172 ......................................................................................22 4.3 Phage Library Screening .........................................................................................................................22 4.3.1 LSm2-3 antibodies from ETH-2 Library ...............................................................................................23 4.3.2 D3B antibodies from Griffin.1 Library ...................................................................................................23 4.3.3 D1D2 antibodies from Griffin.1 Library..................................................................................................24 4.3.4 BSA......................................................................................................................................................24 5. DISCUSSION ...............................................................................................................................................25 6. OUTLOOK....................................................................................................................................................27 7. LITERATURE...............................................................................................................................................28 8. APPENDIX ...................................................................................................................................................30 8.1 Primer sequences and specifications ....................................................................................................30 8.1.1 Primers for D1(78) ................................................................................................................................30 8.1.2 Primers for D2(27-118).........................................................................................................................30 Diploma Thesis Structural Biology Sandra Haenni Paul Scherrer Institute 8.2 Cation Exchange Chromatography ........................................................................................................30 8.2.1 Programs .............................................................................................................................................30 8.2.2 Chromatograms D2D1(78):pRK172......................................................................................................31 8.2.3 Chromatogram D2(27-118)D1:pRK172 ................................................................................................32 8.3 Protocols Phage Display .........................................................................................................................33 8.3.1 ETH-2 Library.......................................................................................................................................33 8.3.2 Griffin.1 Library ....................................................................................................................................35 8.4 ELISA .........................................................................................................................................................42 8.4.1 LSm2-3 ................................................................................................................................................42 8.4.2 D3B.......................................................................................................................................................44 8.4.3 D1D2 .....................................................................................................................................................45 8.4.4 BSA......................................................................................................................................................45 8.5 Buffers and Media ....................................................................................................................................46 8.5.1 Cloning steps .......................................................................................................................................46 8.5.2 Protein Expression...............................................................................................................................47 8.5.3 Cation exchange chromatography.......................................................................................................48 8.5.4 Phage Display......................................................................................................................................48 3 Diploma Thesis Structural Biology Sandra Haenni Paul Scherrer Institute 1. Summary Sm- and LSm- proteins are a protein family whose members are involved in various RNA processing events. They are only functional in form of multimeric complexes, the composition and architecture of which determines the RNA target and function. For the assessment of the role of the individual Sm/LSm-proteins in a given function, tools are required to distinguish first between the individual complexes and then between the individual proteins. Sm- and LSm- proteins show a common fold and share epitopes. Native anti-Sm- autoantibodies from patients suffering from Systemic Lupus Erythematosus as well as most available monoclonal