Protein Expression and Purification Series - Bio-Rad

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Protein Expression and Purification Series • Verify that the desalted sample volume is not >150 µl (assuming two desalting runs using the same column). If the volume is >150 µl, then the column storage buffer was likely not removed properly. DHFR Enzymatic Assay • Ensure the DHF is reconstituted in 10x PBS. • Ensure the NADPH is reconstituted in 1x PBS. • Ensure the DHF and NADPH are used within three to four hours. The reconstituted DHF and NADPH cannot be frozen to retain or extend the activity and shelf life. • The enzymatic reaction occurs very quickly so make sure to add the DHF to the NADPH solution and then place it immediately in the spectrophotometer. It is best to do this step next to the spectrophotometer to ensure no data points are missed. • Make sure the spectrophotometer is turned on at least 30 minutes ahead of time to allow the lamp to warm up. • If using trUView cuvettes, make sure the frosted side is facing the beam path. The frosted side of the trUView cuvettes contains a tiny clear window through which the UV reading is taken. 32 Timeline for the Laboratory Course: Chromatography Instrumentation Purification
Protein Expression and Purification Series Chapter 1: Recombinant Protein Expression and Purification Why Produce Proteins Recombinantly? To be used for research, industrial or pharmaceutical purposes, proteins need to be purified in large quantities. Some proteins, like casein, which makes up 20% of the protein content in milk, can easily be extracted from a readily available source in large quantities. However, most proteins are not naturally produced in a form and in amounts that allow easy purification. The techniques of genetic engineering overcome the limitations of naturally produced proteins by making cells synthesize specific proteins in amounts which can be purified for use in fundamental research or for industrial and therapeutic applications. Biogen, one of the first companies to develop recombinant proteins, is using genetic engineering to produce human interferon beta-1a in Chinese hamster ovary (CHO) cells and is sold under the tradename Avonex. A similar form of recombinant human interferon, interferon beta-1b, is expressed in E. coli and sold by Bayer under the drug name of Betaseron. (An interferon is an immune protein produced in response to a virus, bacteria, parasite, or tumor cell.) Both recombinant human interferon beta-1a and 1b have been developed, tested, and brought to the market to help slow the progression of multiple sclerosis. Without recombinant production of these proteins in CHO cells or in E. coli, there would not be an easy way to obtain this protein for therapeutic usage. Table 1.1 Human proteins produced by genetic engineering. Human proteins produced via genetic engineering and the disease or disorder they are used to treat. Protein Used in the treatment of Insulin Diabetes Somatostatin Growth disorders Somatrotropin Growth disorders Factor VIII Hemophilia Factor IX Christmas disease Interferon-alpha Leukemia and other cancers, MS Interferon-beta Cancer, AIDS, MS Interferon-gamma Cancers, rheumatoid arthritis Interleukins Cancers, immune disorders Granulocyte colony stimulating factor Cancers Tumor necrosis factor Cancers Epidermal growth factor Ulcers Fibroblast growth factor Ulcers Erythropoietin Anemia Tissue plasminogen activator Heart attack Superoxide dismutase Free radical damage in kidney transplants Lung surfactant protein Respiratory distress alpha 1-antitrypsin Emphysema Serum albumin Used as a plasma supplement Relaxin Used to aid childbirth Chapter 1: Recombinant Protein Expression & Purification 33 CHAPTER 1 BACKGROUND
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CHAPTER 3B ADVANCE PREP INSTRUMENTA
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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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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