The overall goal of the following experiment is to obtain a highly purified, full length protein of interest. This is achieved by producing a recombinant bao virus, which is used to infect SF nine insect cells in order to obtain a highly expressed and soluble two tagged version of the protein of interest. A two-step affinity purification is then undertaken, characterized by purification of glutathione, seros tagged protein, followed by metal affinity chromatography.
The result of the purification is a highly pure histidine tagged protein. Next purified protein samples are dialyzed in order to guarantee low salt concentrations in the storage buffer. Results are obtained that show the expression solubility, quantity, and quality of the protein based on whole protein stain of an SDS page gel.
We first had the idea for this method when we had trouble purifying large and unstable proteins such as PAL B two and BOC two. So the main advantage of this technique over existing methods like one step affinity purification, is that in our two step affinity purification protocol, the protein obtained is highly pure and generally free from degradation products. This method can help answering key question in the protein biology field, such as biochemical functions of purified recombinant proteins.
This protocol begins with production of recombinant bao virus, followed by selection of the most efficient BA virus as described in the text protocol for infection. Prepare a spinner culture vessel containing between 0.75 to 1.0 times 10 to the six SF nine cells in 500 milliliters of media with a ratio of one to 150 between virus and media. Place the spinner under a cell culture hood infect the cells by adding the necessary volume of baula virus suspension into the cell suspension through one arm of the spinner.
Let the infected cells grow in suspension at 27 degrees Celsius and harvest the cells when the maximum protein expression is reached, which is determined when choosing the most efficient macular virus. After harvesting cells, lyce SF nine cells expressing the protein of interest in approximately 20 milliliters of GST binding buffer, supplemented with protease inhibitors in an ice water bath. Down the solution 20 times with a down homogenizer then sonicate for three 32nd cycles before dancing again 20 times next, centrifuge the cell lysate at 18, 000 RPM for 30 minutes at four degrees Celsius and keep the supernatant incubate the soluble cell lysate with one milliliter of pre-washed GST beads for one hour at four degrees Celsius under gentle rotation.
The incubation time should be optimized according to the stability of the pro and binding efficiency following binding. Quick spin at 700 RPM and remove supernatant containing unbound proteins. Wash the GST bound proteins with GST washing buffer.
Repeat this process two times after the last wash. Remove as much supernatant as possible. Then incubate the beads with five millimolar A TP and 15 millimolar magnesium chloride in 10 milliliters of GST binding buffer for one hour at four degrees Celsius to avoid non-specific binding of heat shock proteins following incubation.
Wash the beads three times with GST washing buffer. After centrifuging the GST beads and removing the supernatant, wash the GST beads with P five buffer. Next, divide the GST beads, infractions of 100 microliters and add four to eight units of precision enzyme diluted in 100 microliters of P five buffer.
Incubate the GST beads with enzyme for three hours to overnight at four degrees Celsius under gentle rotation. The incubation time should be optimized according to the molecular weight as well as the stability of the protein Following cleavage. Quick spin and collect the supernatant.
Add 100 microliters of P five buffer to the beads. Then repeat, spin, and supernatant collection twice. Pull all the fractions.
Divide the EIT into three fractions of one milliliter and incubate each with 100 microliters of pre-washed talon metal affinity resin dry beads for one hour under rotation, dividing the elution into several fractions, increases binding and washing efficiency. Then wash the resin for five minutes with P 30 buffer at four degrees Celsius with gentle rotation. After repeating the wash two times, pull the beads in a single tube to elute the purified protein.
Add P 500 buffer to the protein bound talon resin. Add a buffer to bead ratio of one-to-one volume.Volume. Incubate the suspension for five minutes under rotation.
Repeat this step twice. Keeping each eluded fraction separate for storage of the purified protein. Dialyze the protein against an appropriate storage buffer two times for one hour at four degrees Celsius under agitation.
It is important to check for precipitation of the purified protein during the dialysis. Make small aliquots of the purified protein and freeze on dry ice for 30 minutes. Store the aliquots at negative 80 degrees Celsius and avoid many thaw freezing cycles.
Analyze the purification process by loading approximately 20 to 40 microliters of each fraction onto an SDS page gel. After running the gel stain with kumasi blue or protein stain for visualization, the soluble and total cell lysates from SF nine cells infected with the REC 14 recombinant valo virus or mock infected as control were analyzed by Kumasi blue staining, allowing confirmation of the expression of GST re 14 his protein during the purification process. Many samples were analyzed to follow the efficiency of the method analysis of these samples by kumasi Blue.
Staining shows that during the first step of purification, GST Rec 14 was correctly bound to GST beads with an apparent molecular weight of approximately 50 kilodaltons due to the fusion of rec 14 with GST after precision cleavage with G-S-T-G-S-T, free rec 14 hiss migrates around 37 kilodaltons. While the cleaved GST can be visualized on the GST beads, despite a portion of GST free REC 14 that still remained bound to GST beads, notable enrichment of REC 14 hiss was achieved. This step could be improved by increasing the incubation time of the protein with precision protease analysis of REC 14 hiss bound to Talon beads compared to the proteins alluded after the GST purification step shows that a large fraction of REC 14 hiss is bound to talon beads.
After several washes, REC 14 hiss was eluded and collected, three illusions were performed. The analysis revealed a high purity of rec 14 hiss and the greatest concentration of rec 14 hiss in the first ellucian comparison of eluded fractions to the talon beads after Ellucian illustrates the efficiency of the illusion. Since little rec 14 hiss can be detected as bound on beads.
These samples were also subjected to Western blood analysis with anti GST and anti hiss antibodies in order to follow REC 14. Throughout the procedure, the anti GST blot shows that some GS TRE 14 and GST alone were present in the alluded proteins from the GST purification step and that contaminants were removed by the hiss tag affinity purification step. This blot reveals that GST had been efficiently removed from re 14 by the precision treatment and the hiss ttag purification step.
The anti hiss blot aims to detect re 14. This blot confirms that GST Rec 14 hiss and REC 14 hiss were not completely cleaved and removed from the GST beads. Moreover, at a high concentration REC 14 seems to aggregate.
In summary, the results presented demonstrate the efficiency of GST hiss purification for the purification of S palm B rec 14. After watching this video, you should have a good understanding of how to recover a highly beautified energy. Combinant protein Once mastered.
This technique can be done in 12 hours after the degeneration of soluble ly acid if it's performed properly. While attempting this procedure, it's important to remember to work quickly and strictly at four degree Celsius to avoid protein degradation Following this procedure. Other methods such as code precipitation assays or mass spectrometry can be performed in order to answer additional questions such as the identification of protein partners or post-translational modification of the protein of interest.
Although we use this method for DNA, repair proteins to strategy could be modified to study the function of proteins in other areas of research.
