Protein Expression and Purification Series - Bio-Rad

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Protein Expression and Purification Series Protein Quantitation SDS-PAGE Analysis Workflow Chapter 8: Purification Protocol for BioLogic DuoFlow System Preparing the BioLogic DuoFlow System for Use Preparing the DuoFlow System and Column Programming the Method Selecting the Fraction Collector Start Method Cleaning the BioLogic DuoFlow System After Use Preparing for SDS-PAGE Analysis Protein Quantitation SDS-PAGE Analysis Workflow Chapter 9: DHFR Enzymatic Activity Protocol Appendices Determining Which Fraction to Test Setting Up Spectrophotometer and Blanking the Instrument Running the Control Reaction Running the Enzymatic Reaction Activity Calculation Workflow Appendix A: Tips and Helpful Hints Appendix B: Results Analysis Appendix C: Determining Your Centrifuge’s RPM Appendix D: Setting up the Bio-Rad SmartSpec Plus Spectrophotometer Appendix E: Setting up a Mini-PROTEAN Tetra Gel Box Appendix F: BioLogic LP System Wiring and Tubing Setup Appendix G: Biomanufacturing: The Production of Protein Pharmaceuticals Using Genetically Engineered Organisms Appendix H: Instructor’s Answer Guide for Focus Questions Appendix I: Glossary Appendix J: References, Legal Notices, and Trademarks 156 158 161 166 167 174 176 178 184 185 187 191 193 196 201 202 203 204 204 205 209 210 213 218 219 223 226 229 234 241 246 6 Table of Contents
Protein Expression and Purification Series Kit Summary Kit Summary The Protein Expression and Purification Series is a modular laboratory course designed to serve four to 12 student teams, depending on which purification option is used. The aim of this course is to express and purify His- and GST-tagged dihydrofolate reductase (GST-DHFR-His) and then analyze the isolated purified protein fractions for enzymatic activity. This project involves growing cultures of GST-DHFR-His, lysing the bacterial cell cultures, separating soluble from insoluble fractions, purifying the GST-DHFR-His via centrifugation or protein purification instrumentation, analyzing protein purity via SDS-PAGE and analyzing protein enzymatic activity. DHFR is an essential protein used in the conversion of dihydrofolate (DHF) into tetrahydrofolate (THF) by the addition of a hydride from NADPH. THF is a methyl (CH 3 ) group shuttle required for synthesis of essential molecules such as nucleotides and amino acids. DHFR deficiencies are extreme and result in symptoms such as megaloblastic anemia and cerebral folate metabolism disorders. DHFR inhibition or reduction disrupts nucleic acid synthesis and cell growth, resulting in cell death. This project offers an opportunity to perform true affinity purification with a realistic research workflow in which students can examine the enzymatic activity of the purified protein product. It also introduces students to the world of biomanufacturing where the research process is scaled up and standard operating procedures and work instructions are followed strictly during the production process. Independent research extensions such as further purification steps, ELISA, Western Blotting, site-directed mutagenesis and many other options are possible once students have purified the protein. The steps in this project to express and purify GST-DHFR-His (and the corresponding chapter in this manual) are: 1. Understanding recombinant protein expression, DHFR, and protein purification (General Background chapter). 2. Setting up the experiments (Instructor’s Advanced Preparation chapters). 3. Growing cultures (Culturing, expression, lysis and SDS-PAGE analysis chapters). 4. Inducing expression of the GST-DHFR-His protein (Culturing, expression, lysis and SDS-PAGE analysis chapters). 5. Lysing the bacterial cells to release the expressed protein (Culturing, expression, lysis and SDS-PAGE analysis chapters). 6. Using SDS-PAGE analysis to verify expression of the protein, identify fractions containing purified protein, and to assess level of purity (Culturing, expression, lysis and SDS-PAGE analysis chapters). 7. Purifing the GST-DHFR-His protein via affinity chromatography followed by desalting of the samples (Purification Protocol chapters). 8. Analyzing the DHFR enzymatic activity (DHFR Enzyme Activity chapter). About This Manual The Protein Expression and Purification Series is accompanied by extensive curricula to enable students to gain a deep understanding about DHFR and the theory behind the techniques used to perform the laboratory series. The manual is provided with the series allowing for the copying and distributing of the entire manual or chosen material to students. Storage Instructions See Checklists for Individual Modules on subsequent pages for shipping and storage conditions for each module. Open each module as soon as it arrives and store components in recommended storage conditions. 7
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX B RESULTS ANALYSIS Protein
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APPENDIX B RESULTS ANALYSIS Protein
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APPENDIX C RCF TO RPM CONVERSTION P
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX E MINI-PROTEAN GEL BOX SET
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX H FOCUS QUESTIONS INSTRUCT
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APPENDIX I GLOSSARY Protein Express
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APPENDIX I GLOSSARY Protein Express
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APPENDIX J REFERENCES, LEGAL NOTES
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Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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