Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography
Trascrizione
For the lysis of harvested E. coli expressing the six-helix bundle SAPN, use a probe with a sonication output of 150 watts, with four seconds of sonication and six seconds of rest to sonicate resuspended bacterial pellets in 40 milliliters of imidazole-free buffer on ice for five minutes.
Centrifuge the cellular lysate to generate a clarified supernatant, and transfer the supernatant to a sterile 150-milliliter flask. Then, bring the sample up to a final volume of 100 milliliters with fresh imidazole-free buffer.
To isolate the protein of interest, open the FPLC software and click the Nuovo Method option. In the Method Settings menu, open the Column Posizione dropdown menu and select C1 Port 3. In the Shown By Technique dropdown menu, select Affinity. In the Column Tipo dropdown menu, select Others, HisTrap HP, and five milliliters. The column volume and the pressure boxes will automatically be set to the appropriate values.
Click Method Outline and drag the Equilibration and Sample Application buttons, three Column Wash buttons, and the Elution button from the Phase Library pop-up menu next to the arrow, before closing the Phase Library menu. Click Equilibration and set the values listed in the table to Initial Buffer B, 4%, Final Buffer B, 4%, and Column Volume, 5.
Click Sample Application. In the Sample Loading box, click the Radio button for Inject Sample on Column with Sample Pump and confirm that the box next to the Use Flow Rate From Method Settings is checked in the Sample Injection with System Pump Box. Change the Volume box value to 100 milliliters and the Fraction Collection Scheme a Enable.
Unclick the Use Fraction Size from Method Settings box and change the fraction size to four milliliters. Click on the first Column Wash button. Set the values listed in the table to Initial Buffer B, 4%, Final Buffer B, 4%, and Column Volume, 10. Click the Fraction Collection Scheme Enable box and unclick the Use Fraction Size for Method Settings. Change the fraction size to four milliliters and click the second Column Wash button.
Set the values in the table to Initial Buffer B, 0%, Final Buffer B, 0%, and Column Volume, 5. Click the Fraction Collection Scheme Enable box and unclick the Use Fraction Size from Method Settings. Change the fraction size to 4 milliliters and click the Third Column Wash button. Set the values in the table to Initial Buffer B, 0%, Final Buffer B, 0%, and Column Volume, 10. Click the Fraction Collection Scheme Enable button and unclick the Use Fraction Size for Method Settings box. Then, change the fraction size to 4 milliliters.
Click the Elution button and right-click the information in the table. In the pop-up menu, click Delete Step, and drag the Isocratic Gradient button onto the table two times so that there are two entries. Set the value for the first entry to Initial Buffer B, 30%, Final Buffer B, 30%, and Column Volume, 10.
Set the value for the second entry to Initial Buffer B, 100%, Final Buffer B, 100%, and column volume, 10. Click the Fraction Collection Scheme Enable button, and click the Use Fraction Size for Method Settings box. Then, click Save As and name the file, Purification.
Place the pump A tubing of the FPLC into the imidazole-free wash buffer, and the pump B tubing into the 500-millimolar imidazole buffer. Place the sample pump tubing into the 100-milliliter sample and run the purification program. When the 60% isopropanol wash is needed, pause the program, move the pump tubing from the imidazole-free wash into the 60% isopropanol wash, and restart the program. At the end of the wash, pause the program again, return the pump A tubing to the imidazole-free wash buffer, and restart the purification program.
