The overall goal of this procedure is to select an optimal HI liquid chromatography column using an automated scouting method for the purification of a recombinant protein with a BioRad DUO flow medium pressure liquid chromatography system. This is accomplished by first preparing requisite buffers and bacterial lysates containing the recombinant protein of interest. Next, the duo flow chromatography system is physically set up, plumbed and prepared for the scouting protocol.
Then the samples are loaded and the programmed HI column scouting method is run to collect the fraction sample eit. Finally, the EIT is analyzed and the optimal hi column and media are identified based on the observed separation characteristics. Ultimately, results can be obtained that show a preferred HI column medium for scaled up purification of the investigator's protein of interest through medium pressure liquid chromatography using the DUO flow automated HI column scouting capabilities.
The main advantage of this technique over existing methods like sequential runs of manual column chromatography, is that automating the process decreases variability and ensures highly reproducible results. Generally, individuals new to this method may struggle due to the physical complexity of modern liquid chromatography systems. However, this can be readily mastered.
Following sufficient practice Demonstrating the procedure will be Orange Stone and Shelly Anderson, two students from my laboratory To start prepare 500 milliliters of the following buffers, 200 millimolar sodium phosphate for buffer, A one 200 millimolar sodium hydrogen phosphate for buffer A two and 4.8 molar ammonium sulfate. For buffer B two deionized water will act as buffer B one degas the buffers to ensure optimal chromatographic operating conditions. The system maximizer will blend together buffers A one and A two to generate a low salt phosphate elution buffer, and it will blend buffers B one and B two to generate the high salt ammonium sulfate.
Start buffer. Prepare 20 milliliters of sample to be loaded by mixing 10 milliliters of cell lysate containing the protein to be purified with 10 milliliters of buffer B two. The final ammonium sulfate concentration of the cell lysate sample solution should be approximately 2.4 molar, making it roughly equivalent to the start buffer.
Filter the lysate buffer mixture through a low protein binding 0.45 micrometer syringe filter to remove particulate matter from the solution. Keep the sample on ice until it is ready to be loaded into the system. The physical setup and plumbing of the chromatography system is one of the most complicated aspects of this procedure.
Set up the duo flow chromatography system according to the diagram shown here. Ensure adequate volumes of buffer are available for the entire protocol and that tubing will continue to remain submerged. Plumb the sample loading valve and dynamic sample loop as illustrated here, connect the sample loop to the sample valve inlet at position three.
Prepare eight identically sized pairs of one 16th inch outer diameter peak tubing. Then connect the pairs to positions one through eight of the two column selection valves. After turning on the UV detector lamps and confirming that all the detectors are calibrated, attach the fraction collector's stream splitter valve and splitter controller as illustrated.Here.
The position two fraction collector splitter valve and splitter controller are operated offline of the chromatography workstation. Set percent split to 10. Replace the standard fraction collector drophead on the position two fraction collector with a microplate drophead.
Rinse the sample loop and flush the chromatography system. Then start a manual one milliliter per minute flow of evolution buffer through the system. Connect one milliliter high trap HI columns to positions two through eight of the column selection valves.
Remove the union connecting the position two column selection valves. Attach the upstream column fittings to the duo flow. Run the elucian buffer through the fittings until all the air is expelled and a large drop of buffer is present.
Next, remove the stopper connected to the column inlet and place a large drop of low salt buffer on the top of the column. Then attach the column to the upstream fittings. Attach the outlet to the downstream column fittings and selection valve tubing.
Set the back pressure limit to 40 PS.I wash the column with five column volumes. Evolution buffer at a flow rate of one milliliter per minute. Wash the column with 10 column volumes of start buffer at a flow rate of one milliliter per minute.
Once the pH UV tracings and back pressure have stabilized manually switched the column selection valves to position one and stop the buffer flow. To load a sample, submerge the sample inlet tubing into the 20 milliliter sample. Then switch the sample loop load wash valve to position one and the sample loading valve to purge.
Initiate action of the sample loop loading pump, drawing up sample into the pump tubing at a flow rate of one milliliter per minute until immediately after sample reaches the sample loading valve. Switch the sample loading valve from purge to load. Restart the sample loop loading pump at a flow rate of one milliliter per minute until 10 milliliters of sample has been drawn into the dynamic sample loop From the setup window on the biologic software.
Select the system devices marked with an asterisk in this table. Next, select six HIC columns to assay program. A linear descending salt gradient and a six column scouting method manually commence the start buffer flow at one milliliter per minute.
Press start on the splitter controller to commence 90%to 10%EIT stream splitting immediately after starting the program method run press start on the onscreen control panel of the offline 96 well plate fraction collector. Observe the realtime display to confirm anticipated run parameters. Compare the GFP specific 397 nanometer absorbance tracing to the general protein 280 nanometer absorbance tracing to estimate relative separation and purification using the different HI media being scouted aliquots of five to 50 microliters from the 96 well plate fractions can be transferred to a fresh plate and assay for specific protein content by Eliza or Western blot and total protein content using SDS page or an Experian microfluidic electrophoresis system representative hick salt gradient conductivity and column pressure of the scouting runs are shown here.
The change in salt concentration as measured by the percentage of buffer drawn from high salt buffer lines is typical of HI methodology. As the salt concentration decreases proteins bound to the column elute conductivity, which corresponds to observed, salt concentration is measured in line immediately following the quad tech and UV detectors. The offset between salt gradient and conductivity tracings indicates the time required for buffer to travel from the buffer inlet through the system and to the conductivity monitor.
Throughout the sample run the system pressure and pH remain relatively constant. This figure shows chromatograms of sequential HI column scouting runs the inline detection of total protein. NGFP is accomplished by measuring the absorbance of light at 280 nanometers and 397 nanometers respectively.
It is possible to approximate the relative GFP abundance and separation for each scouting run. By comparing the two lines in this figure culture tubes for fractions, 10, 12, 14, 16, and 18 of the fennel FF scouting run were visualized under ambient or fluorescent room light and ultraviolet light tubes were viewed both face forward and top down in order to observe the characteristic GFP emission spectrum, GFP is clearly detected in fraction 14. In both UV images, the diffuse blue in the left UV panel is light emitted from the UV lamp.
These post run data corresponds nicely with the inline detection of GFP by measuring eluid absorbance at 397 nanometers, which also identifies fraction 14 as containing the major peak of alluded GFP. Following this procedure. Other methods like western blotting, Eliza's, atomic force microscopy and biochemical reconstitution assays can be performed in order to answer additional questions related to the purity and functional activity of the protein being purified.
While attempting this procedure, it's important to remember the need for manual control of the dual flow system at several points in the method. Examples include buffer refilling and sample loading, checking for air bubbles, and turning on and off detection lamps as necessary. After watching this video, you should have a good understanding of how to use a liquid chromatography system to conduct automated scouting for optimal HI columns and media.
The inline and poster, an evaluated analysis will allow a researcher to select a suitable column for subsequent scale up purification, which may also be combined with other chromatographic methods to increase target protein purity and yield.
