The overall goal of this procedure is to prepare recombinant protein from non pathogenic bacteria to facilitate biophysical chemical studies such as the protein x-ray crystallography or enzymatic kinetics. This method can help answer key questions in how to prepare recombinant protein of interest using non pathogenic bacteria for preliminary biophysical characterization. The main advantage of this technique is that biophysical characterization for protein of interest can be done in any laboratory equipped with common instruments such as cell culture incubators and spectrophotometers.
To begin this procedure, prepare the ligation reaction as outlined in the text protocol. Pipette three microliters of the prepared ligation reaction into a tube containing 50 microliters of chemically competent E.coli DH5 alpha cells. Using a pipette, gently mix the solution.
Ice the solution for 30 minutes. Next, heat shock for 45 seconds at 42 degrees Celsius. After this, add one milliliter of LB medium and grow the cells while shaking vigorously at 250 rotations per minute, at 37 degrees Celsius for 45 minutes.
Spread 100 microliters of transformed cells onto an LB auger plate supplemented with 100 micrograms per milliliter kanamycin. Incubate for 18 hours at 37 degrees Celsius. Using a sterile tip, pick colonies from the plate.
Grow the cells in three milliliters of supplemented LB medium with vigorous shaking at 250 rotations per minute at 18 degrees Celsius for 18 hours. Then, prepare plasma DNA and confirm the nucleotide sequence as outlined in the text protocol. After retransforming the E.coli BL21 strain select a colony using a sterile tip and inoculate 10 milliliters of LB medium containing 150 micrograms per milliliter kanamycin.
Incubate at 37 degrees Celsius for 18 hours with vigorous shaking at 250 rotations per minute. Next, transfer the 10 milliliters of overnight culture to one liter of LB and kanamycin medium. Grow at 37 degrees Celsius until the optical density at 600 nanometers reaches 0.7.
Then submerge the culture in ice cold water for 15 minutes. Add IPTG to the culture, such that the final concentration is one millimolar. Incubate at 18 degrees Celsius for an additional 16 to 18 hours.
After this, centrifuge at 5, 000 times G for 30 minutes at four degrees Celsius. Carefully remove the supernatant. Resuspend the cells in 50 microliters of PBS containing 10 millimolar imidazole.
Next, lyse the cells twice on ice as detailed in the text protocol. Centrifuge at 25, 000 times G for 30 minutes at four degrees Celsius to clear the cell lysate. Then, mix three milliliters of nickel beads and 47 milliliters of PBS containing 10 millimolar imidazole in a 50 milliliter conical tube.
Gravity flow the extra buffer to settle the equilibrated beads in a 2.5 by 10 centimeter glass chromatography column. Using a pipette, transfer the supernatant to the column. Incubate at four degrees Celsius for two hours on a spinning wheel at 20 rotations per minute.
After this, allow the supernatant to drain by gravity. Wash the nickel beads three times using 100 milliliters PBS containing 10 millimolar imidazole for each wash. Then, apply five milliliters of PBS containing 250 millimolar imidazole to elute the RBC 1531 protein.
Analyze 15 microliters of the elution via SDS Page as outlined in the text protocol. Pipette the collected fractions into a dialysis tube. Place the tube into a beaker containing four liters of thrombin cleavage compatible buffer at four degrees Celsius overnight.
The next day, use a pipette to transfer the fractions to a 15 milliliter conical tube. Measure the absorbance at 280 nanometers to estimate the protein concentration as outlined in the text protocol. Next transfer 50 microliters of RBC 1531 protein to several tubes.
Add a different amount of thrombin to each tube and incubate at 20 degrees Celsius for three hours. After determining the least amount of thrombin needed add 10 milligrams of protein and 60 units of thrombin to a 15 milliliter tube. Incubate at 20 degrees Celsius for three hours.
Next, transfer the thrombin digested protein to a centrifugal filter tube. Centrifuge at 3, 000 times G at four degrees Celsius to reduce the volume to less than five milliliters. After this, apply the concentrated protein to a prepared size exclusion chromatography column.
Collect 60 elute fractions with 0.5 milliliters per tube. Run 15 microliters of each fraction on a 15%SDS Page gel as outlined in the text protocol. Then combine the fractions containing the RBC 1531 protein.
To begin transfer the combined fractions to a centrifugal filter tube. Centrifuge at 3, 000 times G at four degrees Celsius to concentrate the protein to approximately 18.5 milligrams per milliliter. Next add 50 microliters of each crystallization screening solution to the wells of a 96 well sitting drop crystallization plate.
Add 0.5 microliters of the concentrated protein solution on a sitting bed. Separately mix the protein solution with 0.5 microliters of the well solution. Cover the plate with clear adhesive film.
Incubate at 18 degrees Celsius and allow for vapor diffusion. Using a light microscope, scan the drops at 20 to 40 times magnification to monitor crystal formation daily for two to three weeks. After preparing the NTP substrates add each to a 96 well plate.
Add 20 micrograms of RBC 1531 protein to each well using a pipette to mix. Cover the plate with its lid and incubate at 37 degrees Celsius for 30 minutes. After this, transfer the plate to a 70 degree Celsius water bath for 15 minutes to stop RBC 1531 mediated catalytic reactions.
Next, add one microliter of S.cerevisiae inorganic pyrophosphatase to each well. Incubate at 20 degrees Celsius for 30 minutes. Then add 16 microliters of molybdate acid solution to the reaction plate to develop the color.
After 15 minutes, read the optical density at 690 nanometers. In this study the protein product of the B serious BC1531 gene is characterized at the molecular level. After purification by nickel affinity chromatography a thrombin digestion trail is performed.
0.3 units of thrombin is seen to be the lowest amount required to completely remove the affinity tag from RBC 1531. After digestion the tag free protein is purified to approximately 99%purity, using size exclusion chromatography. Then, the oligomeric status is estimated by size exclusion chromatography.
Because RBC 1531 is eluted at approximately 84 milliliters its molecular weight is estimated to be 55 kilodaltons. The monomer's calculated molecular weight is about 13 kilodaltons. Therefore, RBC 1531 assembles as a tetramer.
Crystallization screening is then performed utilizing a sitting drop vapor diffusion method. Crystals are seen after two days with one molar sodium citrate and 20%polyethylene glycol 3, 000, and with 0.1 molars sodium cacodylate and one molar sodium citrate. After this NTPase activity is determined.
No visible color change is seen after adding molybdate to the RBC 1531 mediated hydrolysis reaction. However, when pyrophosphatase is added the color is seen to change to deep blue. The optical density at 690 nanometers and NTP concentration are then analyzed using non linear regression.
The RBC 1531 enzyme is determined to have a maximum velocity at 0.75 and a Michaelis constant of 10 micromolar. While attempting this procedure it's important to prepare proteins samples without delay and to discard fractions containing any impurities that impede protein crystallization or introduce errors in enzymatic assays. After its development this technique paved the way for researchers in the field of enzymology, and other protein science to characterize proteins of interest with spectrophotometers that are decorated in most laboratories.
Though this method can provide insight into general ideas of recombinant protein preparation, crystallization screens, and enzymatic kinetics, it can also be applied to biophysical chemical analysis for other proteins of our interest. Don't forget to be careful when working with proteins as they can be extremely unstable. Precautions such as leaving samples over the ice and refrigerating at 80 degrees for long term storage will be desired.
After watching this video you should have a good understanding of how to prepare recombinant protein from non pathogenic bacteria to obtain protein crystals for a structure determination by x-ray crystallography and how to characterize enzymatic activity using a colorimetric method.
