The overall goal of this procedure is to reconstitute the Trium Castine telomerase in vitro and to determine its activity. This is accomplished by first expressing recombinant his tag, Trillium Castine turt, or TC turt protein in e coli. The expressed TC turt protein is then purified through various chromatography steps.
The next step of the procedure is to culture the trium Castine beetles and to isolate the total RNA from trium castane beetle larvae. The telomerase enzyme is then reconstituted in vitro by mixing recombinant TC turt with the extracted total RNA. Ultimately, results can be obtained to show that in vitro telomerase reconstituted from recombinant protein and total RNA produces an act of telomerase enzyme as visualized in the trap assay.
Though this method can provide insight into the trium KE telomerase, it can also be applied to other systems such as other protein nucleic acid complexes. Generally, individuals new to this method will struggle because isolation of the beetle total RNA is extremely sensitive to RNA degradation and therefore great care must be taken in order to ensure proper reconstitution. This protocol begins with the expression of recombinant TC TURP with the tobacco etch virus protease Cleavable N terminal HEXA histamine tag in transformed Rosetta DE three P lyse S cells as described in the written protocol Following cell harvest, sonicate the cells on ice for two minutes in 32nd intervals with 32nd pauses at approximately 51 watts of power.
Spin down the lysed cells at 18, 000 RPM for 20 minutes to separate the soluble and insoluble protein fractions and carefully remove the SUP natin. Purify the supra natin using nickel affinity chromatography according to the written protocol. ELUTE TC turt off the nickel NTA resin using an ole gradient and collect three milliliter fractions.
For SDS page analysis, use page analysis to determine which fractions contain the TC turt protein. Concentrate those fractions in an AmCon 30 kilodalton Millipore filter to a volume less than 10 milliliters. Next pre equilibrate a HS porous column.
To further purify the TC Turt protein by cation exchange chromatography, load the protein mix on the HS porous column and wash it To remove all protein and nucleic acid contaminants. Continue to wash the column with buffer containing 500 millimolar potassium chloride, followed by 700 millimolar potassium chloride to remove any remaining contaminants. Elute the TC Turt protein off the HS porous column with one molar potassium chloride buffer.
At this point, the protein should be more than 99%pure. Finally, remove any protein aggregates by passing the protein over a pre equilibrated SUEx 200 size exclusion column. The purified TC Turt protein is now ready for in vitro reconstitution.
Several weeks prior to this protocol, the Trium Castin culture was started by adding 100 adult beetles to a container with 28.5 grams of flour and 1.5 grams of dried baker's yeast. The cultures were stored at room temperature in a dark, humid environment for three to four weeks to allow for reproduction and growth. Following incubation, harvest 20 beetle larvae from the culture by separating them from the adult beetles and the flower yeast culture.
Eliminate flower or yeast particles attached to the beetles before grinding them. Sterilize the bench area and the mortar and pestle with RNA zap to remove all traces of ribonuclease before grinding the beetles in liquid nitrogen. Grind the larvae into a fine powder using the mortar and pestle.
Transfer the powder to a 50 milliliter centrifuge tube while maintaining the liquid nitrogen to ensure that the powder stays frozen until the extraction buffer is added. Allow excess liquid nitrogen to evaporate from the powder after the liquid nitrogen has evaporated. Homogenize the beetle powder in 200 microliters of extraction buffer and incubate on ice for 30 minutes.
Centrifuge the homogenate at 15, 000 RPM for 20 minutes at four degrees Celsius. Following centrifugation flash freeze the collected supernatant in liquid nitrogen. Finally extract total RNA using the kyogen RN easy mini minikit.
Begin in vitro reconstitution of T castin telomerase by mixing 20 micrograms of recombinant hist TAG TC TURP protein with 50 microliters of T castin total RNA in binding buffer incubate the TC turt total RNA mixture for two hours at 22 degrees Celsius in T castin lysate, and the presence of RN aen inhibitor to prevent RNA degradation. Finally, purify the complex over a nickel NTA column to remove lysate impurities and excess RNA. The reconstituted t castin telomerase is now ready for the telomerase repeat amplification protocol assay.
To begin the trap assay, mix four micrograms of nickel NTA purified T castin telomerase in 50 microliters of elongation buffer. Incubate the mixture at 30 degrees Celsius for 60 minutes. To allow elongation of single strand DNA by the reconstituted T castin telomerase.
Extract the elongated single stranded DNA by adding an equal volume of phenol chloroform to the reaction mixture. Shake the mixture gently until the emulsion is homogeneous. Centrifuge the sample at 12, 000 RPM for five minutes at four degrees Celsius to separate single stranded DNA from contaminants.
Transfer the top aqueous layer of the sample to a 1.5 milliliter einor tube. After adding sodium acetate pH 5.0, magnesium chloride and ethanol, allow the sample to precipitate overnight at minus 20 degrees Celsius the following day. Spin the sample at 15, 000 RPM for 30 minutes at four degrees Celsius to pellet the precipitated single stranded DNA.
Remove the solution using a pipetter and then add 200 microliters of 70%ethanol to the sample to wash out the remaining sodium acetate and magnesium chloride. Centrifuge the sample for five minutes at the same speed and temperature to repel the sample pipe. Head off the ethanol solution and dry the remaining traces of ethanol by allowing the einor tube to incubate open at room temperature for three minutes.
Having ethanol present in the sample will inhibit PCR amplification After the ethanol dries, resus suspend the single stranded DNA pellet and 50 microliters of PCR reaction buffer. Then PCR amplify the telomerase elongation products following amplification. Pre-run a 12%poly acrylamide gel with tris bore eight EDTA running buffer for 30 minutes.
After pre-running and loading the gel with the PCR reactions, run the gel at 185 volts for 1.5 to two hours. As a final step, dry the gel and use the phosphor imaging plate to visualize activity shown. Here is an example of purified TC turt after size exclusion chromatography.
The protein concentrated after purification is run on a 12%SDS poly acrylamide gel and is shown to be more than 99%pure a size exclusion. Chromatogram is also provided to demonstrate the purity and lack of aggregates from the purified protein sample. Note that the final protein yield can vary.
Although an expected yield is five to 10 milligrams, after all purification steps have been completed, A representative trap assay for the reconstituted T castin telomerase is shown. The stepwise ladder of telomerase products can be seen in lane one. However, they're absent in both the RNAs treated telomerase and the turt active site mutant samples in lane two and three respectively.
After watching this video, you should have a good understanding of how to reconstitute the telomerase enzyme in vitro using recombinant protein and total RNA as well as be able to test for telomerase activity While attempting this procedure, it's important to remember to treat all samples, especially the extracted total RNA with great care since any R a's contamination will yield no result.
