Protein Expression and Purification Series - Bio-Rad

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APPENDIX H FOCUS QUESTIONS INSTRUCTORS GUIDE Protein Expression and Purification Series Appendix H: Instructor’s Answer Guide for Focus Questions Focus Questions: DHFR Cancer Connection 1. 2. 3. 4. What is DHFR? Why is it important? DHFR, or dihydrofolate reductase, is an enzyme that converts dihydrofolate into tetrahydrofolate. DHFR is an essential enzyme in the synthesis of purines, thymidylic acid, and aminio acids. What role does DHFR play in cancer? Inhibited DHFR leads to a shortage of thymidylates, which interferes with nucleic acid synthesis, leading to a lack of cell growth and proliferation. Because cancer involves uncontrolled cell growth and proliferation, a lack of DHFR activity slows down or stops cell growth and proliferation. How does methotrexate interfere with DHFR? Methotrexate is a folic acid mimic that interferes in folic acid metabolism. Without folic acid, DHFR cannot catalyze the reaction to create thymidylates necessary for DNA synthesis. Methotrexate is a competitive inhibitor of DHFR. It can bind to the DHFR and block the binding of DHF. What reaction does DHFR catalyze? What is the cofactor that is necessary for this reaction to occur? DHFR catalyzes the conversion of DHF to THF. It reduces a double bond. NADPH is the cofactor that is required for this reaction to occur. 5. Name an inhibitor of DHFR. How does this inhibitor affect its action? Methotrexate. Methotrexate has a structure similar to dihydrofolate (DHF). It acts as a competitive inhibitor since it can bind to the active site and prevent binding of DHF. If DHF cannot be bound in the active site by DHFR then it cannot be converted to THF. 234 Appendix H: Focus Question Answers
Protein Expression and Purification Series Focus Questions: Protein Expression 1. 2. 3. 4. What is a recombinant protein, and why would it be used instead of a native protein? A recombinant protein is a protein produced from genetically engineered, or recombinant, DNA. The DNA sequence that codes for a particular protein is inserted into a plasmid, which is inserted into a cell, which in turn produces the protein of interest. A protein would be produced recombinantly if it could not be purified from natural sources in large enough quantities to be useful for research or pharmaceutical use. Name at least one pro and one con of using eukaryotic cells to produce a recombinant protein. When would you choose to use eukaryotic cells to produce a recombinant protein? Pros: Eukaryotic cells perform post-translational modifications such as glycosylation, can refold complicated proteins, and can secrete recombinant proteins into the medium for easier purifications. Cons: Eukaryotic cells are slow to grow and can be expensive to grow and maintain. Eukaryotic cells would typically be chosen for large or complicated proteins, and when posttranslational modifications are needed for proper protein function. Name at least one pro and one con of using prokaryotic cells to a produce recombinant protein. When would you choose to use prokaryotic cells to a produce recombinant protein? Pros: Prokaryotic cells are fast growing and inexpensive to culture and grow. They can produce large quantities of recombinant protein relative to their total protein content. Cons: Prokaryotic cells do not perform post-translational modifications and many times express recombinant proteins in an insoluble form. This can make purification of a refolded active form difficult. Prokaryotic cells would typically be chosen when the recombinant protein does not require posttranslational modifications and when it can be expressed solubly or easily refolded. What are three considerations for recombinant protein gene design? Three considerations for recombinant protein gene design are: 1, coding efficiency of the gene; 2, the rate at which the protein will be expressed; 3, how the recombinant protein will be purified away from host cell proteins. 5. What is the log phase of a cell culture, and why is it important to recombinant protein production? During log phase the bacteria are actively growing and dividing, producing more cells. For recombinant protein production, it is important that the cells are healthy and actively growing in order to use their cellular machinery to produce protein. 6. 7. Why are subcultures prepared when trying to produce recombinant proteins? It is hard to predict exactly when cells will be in log phase when grown from a single colony so an initial culture is prepared and used to start a subculture. What is the purpose of adding glucose to culture media for lac operon systems? The lac operon is repressed when no lactose is present and if glucose levels are high. LB medium is made with casein hydrolysate and yeast extract both which may have endogenous lactose. The addition of glucose ensures the repression of gene expression so that the recombinant protein production is tighly controlled. Appendix H: Focus Question Answers 235 APPENDIX H FOCUS QUESTIONS INSTRUCTORS GUIDE
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Biotechnology Explorer TM Protein E
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Protein Expression and Purification
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CHAPTER 1 BACKGROUND Protein Expres
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CHAPTER 2 FOCUS QUESTIONS Protein E
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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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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Page 184 and 185: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 202 and 203: CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
Page 210 and 211: APPENDIX A TIPS AND HELPFUL HINTS P
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Page 214 and 215: APPENDIX B RESULTS ANALYSIS Protein
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Page 218 and 219: APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
Page 222 and 223: APPENDIX D SETTING UP THE SMARTSPEC
Page 224 and 225: APPENDIX E MINI-PROTEAN GEL BOX SET
Page 226 and 227: APPENDIX F BIOLOGIC LP SYSTEM SETUP
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Page 230 and 231: APPENDIX G BIOMANUFACTURING Protein
Page 232 and 233: APPENDIX G BIOMANUFACTURING Protein
Page 236 and 237: APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 242 and 243: APPENDIX I GLOSSARY Protein Express
Page 244 and 245: APPENDIX I GLOSSARY Protein Express
Page 246 and 247: APPENDIX J REFERENCES, LEGAL NOTES
Page 250: Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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