Protein Expression and Purification Series - Bio-Rad

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APPENDIX A TIPS AND HELPFUL HINTS Protein Expression and Purification Series Appendix A: Tips and Helpful Hints How to get the best protein expression and purification results 1. Culture, subculture, and induction The time frames listed in the instruction manual are designed to have as much flexibility as possible to allow for classroom time periods. However, when possible, the following guidelines will help you achieve the best results for cell growth and protein expression. • Initial overnight cultures should be between 14-20 hours rather than 24 hours. • Induction levels are the highest when induction occurs for four hours. The figure below shows a comparison of cells induced for four hours versus 24 hours, demonstrating the clearer induction of the cells induced for four hours. 250 kD 150 100 75 50 37 25 20 15 10 1 2 3 4 Figure 1. Gel comparing four-hour induction to 24-hour induction. The four-hour induction lane has a much more defined band of GST-DHFR-His (lane 3, arrow) relative to all of the background E. coli proteins when compared to 24 hours of induction (lane 4, arrow). Lane Sample 1 Precision Plus Protein Dual Color standards 2 Uniduced cells 3 Cells induced 4 hours 4 Cells induced 24 hours 2. Stability of induced protein While the BL21(DE3) E. coli are deficient in some proteases, extra protease inhibitors are not being added to the cell lysate due to chemical safety. • After induction of the BL21(DE3) cell culture, the best stopping point for protein stability is after pelleting the cells and removing the supernatant to form what is called a cell paste. • This cell paste is highly stable when stored at –20°C or –70°C. 3. Lysis of cells The most efficient lysis of cells requires the cells to be completely resuspended followed by complete freezing and thawing of the resuspended cells. • Completely resuspend the cell paste in the lysis buffer containing lysozyme. This may require vortexing and vigorous pipeting. • An ethanol/ice bath is the preferred method of freezing. • Thaw frozen lysed cells completely before beginning each subsequent freeze-thaw cycle. • If an ethanol/dry ice bath cannot be used, the freeze-thaw can be accomplished in a –70°C freezer overnight or by two cycles of overnight freezing followed by thawing using a –20°C freezer. A –20°C 210 Appendix A: Tips and Helpful Hints
Protein Expression and Purification Series freezer is the least preferred method of lysing cells. When using the –20°C freezer method, it is imperative that the cell pellets are completely frozen to achieve lysis of the cells. If the resuspended cell paste is not completely frozen, adequate lysis will not occur. 4. Separation of soluble and insoluble fractions A centrifuge capable of exerting 16,000 x g of force is necessary to separate the soluble and insoluble fractions. Since no DNase is used in this protocol, the insoluble fraction and genomic DNA tend to form a viscous blob rather than a defined pellet. Care is needed to not transfer the genomic DNA into the soluble fraction when separating the soluble and insoluble fractions. • Spin the lysate at 16,000 x g for 20 minutes. For a Bio-Rad Model 16K microcentrifuge, this is comparable to 14,000 rpm. For different instruments, please refer to the instrument's instruction manual and refer to Appendix C for help in determining the appropriate speed to use. • Extreme care is needed to not decant the insoluble fraction. If it is decanted into the soluble fraction, carefully try to separate the fractions again. If this is not possible the lysate can be centrifuged at 16,000 x g for another 20 minutes to re-separate the fractions. • If genomic DNA carries over to the soluble fraction and is too viscous, the DNA can be sheared with a 22 gauge needle to break it up for easier handling in subsequent steps. The figure below shows the insoluble and soluble fractions and the relative amounts of GST-DHFR-His found in each fraction. 250 kD 150 100 75 50 37 25 20 15 10 5. Affinity purification—Centrifugation methodology A variable speed microcentrifuge is required for the affinity purification. It is critical to set the variable speed microcentrifuge so that it generates 1,000 x g of force. During the affinity purification, it is important to perform each step in the correct order. • For the Bio-Rad Model 16K microcentrifuge, 1,000 x g is equivalent to a speed of 3,500 rpm. A Bio- Rad mini-centrifuge should NOT be used since these produce 2,000 x g of centrifugal force which is too much for the Ni-IMAC resin to withstand. • Make sure to mix the slurry of Ni-IMAC resin fully to resuspend the chromatography beads. • It is extremely important to make sure that once the column is capped on both ends, that the resin and soluble fraction are thoroughly mixed before putting the column on a tube roller or rocking platform. Appendix A: Tips and Helpful Hints 1 2 3 4 5 Figure 2. Gel analysis of recombinant GST-DHFR-His expression and location of GST-DHFR-His after lysis. Lane 3 shows a strong band of induced GST-DHFR-His expression (at arrow). Lanes 4 and 5 show comparable levels of GST-DHFR-His in the soluble and insoluble fractions. Lanes 4 and 5 also have a prominent lower band near 14 kDa. This is the band for lysozyme (which is a good way of verifying that students added lysozyme to their samples). Lane Sample 1 Precision Plus Protein Dual Color standards 2 Uniduced cells 3 Cells induced 4 hours 4 Insoluble fraction 5 Soluble fraction 211 APPENDIX A TIPS AND HELPFUL HINTS
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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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Page 160 and 161: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 202 and 203: CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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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
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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
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Page 234 and 235: APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 242 and 243: APPENDIX I GLOSSARY Protein Express
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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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