This video demonstrates a novel single step fret assay for quantitative analysis of protease kinetics. The general strategy for the following assay is based on fret signal analysis. Here, the fret pair, cype and ype are attached to the N and C termini of pre sumo one in the intact substrate and cype and ype are enclosed proximity.
When this molecule is excited at 414 nanometers, the majority of the emission signal is at 530 nanometers. However, when SENP one protease is added, the substrate is cleaved at the Gly glyc in Sumo one C Terminus separating CIPA and wpa. Now when excited at 414 nanometers, the majority of the emission signal seen is at 475 nanometers.
To assess the kinetics of this reaction, fluorescence emissions are determined every 15 seconds over a period of five minutes. Analysis of the resulting fluorescence data reveals the rate at which the cype pre sumo one ype substrate is digested by the SENP one protease. The main advantage of this technique over existing mass, like a fat assay using fluorescence peptide substrate or radi metric analysis or western blood, is that this technique can determine the realtime kinetic parameter in a single step realtime assay.
Implications of this technique extend towards therapy and diagnosis of proteins involved diseases such as cancers and the metabolic disease. Because this technique can provide precise and high throughput assays for various pros and their inhibitor discovery and the characterizations demonstrative disease procedure will be ENUA grad student from my laboratory, who was the first person to develop this method and procedure. Begin this procedure by amplifying the open reading frames of the Cype ype pre sumo one, mature sumo one, and the SENP one catalytic domain by PCR.
Clone the resulting products into PCR two topo vector. Next, confirm the sequences of the genes are correct by sequencing. Then clone the CD NA encoding CYPE pre sumo one ype cype sumo, one ype, and catalytic domain of SENP one into the PET 28 B vector with an N terminal Polyhis tag.
After the genes have been cloned into the vector, transform them into Esia coli, BBL 21 DE three by electroporation. After plating the transformed bacteria on solid medium, choose single colonies and culture them overnight. Next, inoculate two liter flasks of two XYT medium with the transformed bacteria at a ratio of one to 100.
Incubate the cultures for three hours at 37 degrees Celsius, shaking at 250 RPM to reach an optical density of 0.5 at 600 nanometers. To induce protein expression, add IPTG to a final concentration of 100 micromolar and shake for 16 hours at 25 degrees Celsius at 200 RPM. Next, transfer the bacteria to 250 milliliters centrifuge bottles.
Then harvest the bacteria by centrifugation at 8, 000 G at four degrees Celsius for five minutes. Following the spin, discard the supernatant and resuspend the bacterial cells in a lysis buffer of tris HCL sodium chloride and ole transfer the cultures into 50 milliliters. Centrifuge tubes then sonicate the cells at an amplitude of 70 for 10 minutes at five second intervals to break up the bacterial cells.
Then to separate fusion proteins from cell debris centrifuge the tubes at 35, 000 RCF at four degrees Celsius for 30 minutes. During the spin, set up a 10 milliliter BioRad column with 500 microliters nickel NTI beads for each liter of cultured cells. Let the solution drip from the column following the spin.
Transfer the supernatant into a new 50 milliliter conical tube. Pour the supernatant solution into the column. Once all of the supernatant has entered the resin, wash the column twice with 10 milliliters of a tris hydrochloride sodium chloride, and emit aole buffer.
To elute. The protein add 500 microliters of the same buffer this time containing 500 millimolar ole. Collect the flow through in a micro centrifuge tube.
Next, transfer the solution to dialysis tubing with a molecular cutoff of 10 kilodaltons and dialyze the sample overnight in one liter of buffer containing 20 millimolar tris, HCL 50, millimolar sodium chloride, and one millimolar DTT at four degrees Celsius. Following dialysis, transfer the purified proteins to a micro centrifuge tube, determine the concentrations of the purified proteins by Bradford Assay. Next, the purified proteins are used to assess the rate at which pre sumo one is cleaved by SENP one using a quantitative fret assay.
In micro centrifuge tubes prepare 60 milliliters of Spet Sumo one and YA at concentrations of 5100, 200 500, 750, and 1000 ano molar. These control samples will be used to characterize the constant ratio of direct emission of cype and YA in the same buffer. Prepare cype pre sumo one y PET at 12 concentrations ranging from 0.03 micromolar to 0.9 micromolar and a 0.3 nano molar dilution of the catalytic domain of SENP one.
Set up the fluorescence multi-well plate reader to measure the fluorescent emission at 475 and 530 nanometers after an excitation at 414 nanometers and emission at 530 nanometers after an excitation at 475 nanometers at 32nd intervals for two minutes at 37 degrees Celsius. To obtain autofluorescence of the plate, place an empty 384 well plate in the fluorimeter and take the measurements. Once the autofluorescence of the plate has been determined, adjust the settings of the fluorimeter so that for Cype Sumo one excitation will be 414 nanometers and measurements will be taken at 475 nanometers and 530 nanometers for ype excitation will be 414 nanometers and 475 nanometers and measurements will be taken at 530 nanometers.
Next, add 75 microliters of cype Sumo one ype or cype sumo one ype to the appropriate wells of the 384 Well plate in triplicate as shown in this diagram. Then add five microliters of the prepared SENP one to each well bringing the final volume to 80 microliters immediately place the plate in the fluorimeter and begin the fluorescence measurements following the run. Analyze the fluorescent readings using the quantitative fret analysis method demonstrated in the next section.
The overall idea of this technology is to correlate the absolute threat signal with the digestion. Want to get the absolute threat signal? We need to exclude the direct contributions of donor and the acceptor from the total fluorescent emission at acceptors emission wavelengths.
The direct contributions of donor and the acceptor are calculated from the correlation coefficient factors arfa and the beta of donor and the acceptors respectively. In this section of the video will detail how the calculations are made. To analyze the kinetics of substrate digestion by SENP one, use GraphPad Prism five software begin by opening the exported txt file in Excel after subtracting the background fluorescence of the plate.
The digested substrate concentration at different time points is calculated by PrepU formulations when excited by light of wavelength 414 nanometers, the total fluorescence emission of spet pre sumo one ype at 530 nanometers can be derived from three sources, the absolute fret induced Y pets, emission, cype, direct emission, and wpe direct emission. In this equation, FL five 30 slash four 14 is the total fluorescence emission at 530 nanometers. When excited at 414 nanometers, FL fret is the absolute fret signal.
FL Spet Con is the Spet direct emission. When excited at 414 nanometers and FL Ype Con is the Ype direct emission. When excited at 414 nanometers, the direct emission of spet at 530 nanometers is proportional to its emission at 475 nanometers.
When excited at 414 nanometers with a constant ratio of alpha, the direct emission of YA at 530 nanometers under excitation at 414 nanometers is proportional to its emission at 530 nanometers. When excited at 475 nanometers with a constant ratio of beta when spet pre sumo one ype is digested by SENP one and excited at 414 nanometers. The fluorescent mission at 530 nanometers is decreased, but can still be divided into three parts as here.
FL Prime five 30 slash four 14 is the total fluorescence emission at 530 nanometers after digestion. When excited at 414 FL Prime fret is the remaining absolute fret signal and FL prime SPET 4 75 slash four 14 is the spet emission at 475 nanometers after digestion when excited at 414 nanometers. The emission is from two parts undigested cype, pre sumo, one ype, and digested cype Sumo one.
An FL Y PET five 30 slash 4 75 is the ype emission when excited at 475 nanometers, which is constant whether cype pre sumo one ype is digested or not after digestion by SENP one, the remaining fret emission is shown in this equation where C is the total concentration of cype. Pre sumo one ype, and X is the concentration of digested cype. Pre sumo one YA.By combining all of the items, the detected fluorescence emission at 530 nanometers under excitation of 414 nanometers is shown by this equation for the fret based protease assay for enzyme kinetic study, the reaction rate is correlated with the change in the amount of substrate as the concentration of product increases exponentially from zero.
When T equals zero as seen here, S subzero indicates the original substrate concentration when T equals zero. The original velocity is maturation of pre sumo. One by SENP.
One can be determined by monitoring the changes in the fluorescent signal at 475 and 530 nanometers during the process. As shown here, the velocity of pre sumo one's maturation was substrate dose dependent, which suggests that the catalytic domain of SENP one shows excellent activity for presum O one's maturation. The initial reaction velocities were calculated using the same analysis with different substrate concentrations.
The KCAT to KM ratio is generally used to compare the efficiencies of different enzymes with one substrate or a particular enzyme with different substrates. KM and Vmax can be obtained from the McKayla Cementin equation by plotting the various initial velocities corresponding to the different concentrations of cype. Pre sumo one YA kcat was obtained using this equation.
According to the above analysis, the calculated KM was 0.21 plus or minus 0.4 micromolar. The kcat was 6.90 plus or minus 0.28 per second, and the kcat km ratio was 3.2 plus or minus 0.55 times 10 to the seventh per molar. Second, After watching this video, you should have a good understanding of how to determine protease connect as parameter using a novel quantitative fat assay in a single step measurement in real time following this procedure.
Other method, like a product inhibition or inhibitor characterizations can be performed in order to answer additional questions like real apparent KM or inhibitor K.This technique paved the way for researchers in the field proteins to explore accurate genetics, understanding of proteins involved physiological and the pathological process in optical sensor. Immediate diagnosis and drug discovery.
