Investigating the Effects of Mutation on Active Site Amino Acids - Lab Report Example

?Intro The synthesis of altered proteins with new binding sited, designed to target specific groups of molecules, is one of the major tasks of modernprotein engineering. Significant efforts in this area were highlighted by limitations of natural proteins in both biomedical and analytical applications. Generally, there are two ways of producing modified proteins. Usually, turns and loops are the first areas of interest while considering engineering of new binding sites. Modifications are done either by creating new binding sites introducing random mutations on the surface of strands, helices or randomization of already existing regions that connect secondary structures. Using these two techniques it is possible to synthesize a protein that will bind any desired target. As recent studies suggest, it is possible to add random peptide sequences into loops of ?-lactamase subsequently establishing the catalytic properties of the produced ?-lactamase derivatives. The same authors highlighted the fact that there is no correlation between tolerance to insertion and tolerance to mutagenesis. A turn between two ?-strands next to the active site was found to be inactive in random mutagenesis but demonstrated the opposite in insertions. The present work consists of three elements. Initially it is creating a construct (cloning a mutated gene into an expression vector) ?-lactamase a. using traditional cloning methods (overlapping PCR for mutagenesis, digestion, ligation). Then move on to Protein- Prep- expressing and isolating mutated ?-lactamase a, transformation of construct into competent cells b and protein purification by GFC and IEC before, finally, move on to investigating the effects of mutation on the functionality of ?-lactamase a. Activity assay of mutants compared to those of the WT enzyme A Procedure Week 1: PCR- Primer Design/PCR Mutagenesis Two sterile 0.2 ml PCR tubes were loaded with 5 µL PFU buffer, 3 µL dNSO, 2.5 µL template, 0.5 µL PFU, 26.5 µL H2O each. Also, one tube was loaded with 5 µL Reverse Primer and 5 µL Forward Primer Mutant while the other was loaded with 5 µL Forward Primer and 5 µL Reverse Primer Mutant. 23 cycles of PCR were used to generate the required amount of the DNA sequence of interest. Denaturation, annealing, and elongation represent one cycle of PCR. The first minute of DNA generation was conducted at 950C the second at 500C. The temperature for the following three minutes was raised to 720C with subsequent 10 minutes of elongation at 720C before finally cooling down to 40C affording the crude product. Week 2: PCR Fragment Purification and Restriction Digest A. The crude product produced on the previous stage was loaded into the wells of 0.4 % agarose gel, the first run was conducted. All bends were cut and 330 µL QG buffer was added. The mixture was heat till the gel dissolved completely after that transferred to the column and span for 2 minutes. 500 µL QG buffer was added and spinning was continued for extra three minutes. 30 µL EB buffer was added to dissolve DNA and spinning was continued for 2 minutes. In this way DNA was pulled through. B. To generate the required amount of DNA PCR was conducted. Each of the two sterile 0.2 ml PCR tubes were loaded with 5 µL PFU buffer, 5 µL Forward Primers, 5 µL Reverse Primers, 2.5 µLdNTP, 0.5 µL pfu, µL H2O. Also, one tube was additionally loaded with 5 µL AB DNA (Forward mutant) while the other 5 µL CD DNA (reverse mutant). On the next day the first tube was loaded with DNA 30 µL, Eco R1 buffer 4 µL, Eco R1 - 1 µL, Hind III- 1 µL, H2O- 4 µL and the second with 4 µL vector, Eco R1 buffer 4 µL, Eco R1- 1 µL, Hind III- 1 µL, H2O- 4 µL. Both tubes were left at 370C overnight. Week 3: Restriction Fragment Purification/Ligation/Agar Plate Preparation The gel run was initiated following purification of the previously generated DNA samples. DNA concentration was measured and was found to be 5 µL into 500 µL. The following ligation was conducted. The ratio PCR/Vector was 3/1 Week 4: DNA Transformation/ Colony Screening (DNA Isolation and Restriction Digest) All the DNA material was added to the obtained cells and everything was put in ice for 15 minutes. 250 µL LB medium was added. The mixture was shooke for one hour following centrifugation for 2 minutes. 200 µL medium was removed using a pipet to w/o disturb cell pallet. Cells were resuspended in what was left of the medium. Dispension onto agar plate were used to finish DNA Transformation/ Colony Screening. Week 5: ?-Protein purification, Dialysis lactatamase expression and purification DNA construct of each of the produced mutations (PET21 vector containing the mutant) was transformed into BL21(DE3) cells to express the protein. The used cells were grown in Louria broth (LB) containing amphicilin plates for 16 hours at 370C. Inoculation of a single colony of each mutant in 50 ml of Louria broth (LB) medium was conducted in the presence of ampicillin (100 µL of amp/ 1mL LB ratio) and grown overnight. On the following day, 50 mL culture was added to 500 mL of LB media and 50 mg of amphicilin was added to it . The culture was grown in 370C and when cell density reached 0.8 O.D. expression was induced by 1 mM IPTG for 4 hours at 370C. After four hours of expression the cells were removed at 6000 rpm for 8 minutes. The supernatant was poured off and pellet was kept at -200C. On the following day the pellet was resuspendet in 15 mL of the previously produced osmotic shock buffer (20 mM, Tris, pH 8.0 containing 0.16M Nack and 0.2 mM Sucrose). After the complete resuspension of the pellet, centrifugation of the mixture against a balance was conducted in the small centrifuge tube for 15 minutes at 15.000 rpm and the supernatant was collected. The final solution was subjected to dialysis against 20 mM Tris, pH 8.0 buffer for 16 hours to completely remove sucrose. Week 6: Gel filtration chromatography Once the column had been filled approximately one third of capacity with elution buffer, a valve (near the lower portion) was released, allowing the solution within to be slowly channeled. The pipette was then used to add another solution (dilute slurry) to the matrix material within the column. This solution was stirred throughout this step, and was continually mixed in until the desired height was obtained. The flow was stopped by closing the valve, and gently the sample was gently added against the wall of the column without disturbing the bed. After the addition, the valve was opened and the sample was allowed to flow into the bed. At this point the valve was closed and a small amount of elution buffer was gently added. This procedure was repeated with the buffer and finally a larger quantity of the buffer was added without disturbing the bed. One millilitre (1 ml) from each sample was placed into the test tubes, and held until total elution. The results bellow were measured at 280nm: Results: Fractions Absorbance 1 0.495 2 0.345 3 0.321 4 0.264 5 0.237 6 0.360 7 2.677 8 2.791 9 2.627 10 2.517 11 2.434 12 1.972 13 1.549 14 1.366 15 0.946 16 0.841 17 0.737 18 0.638 19 0.607 20 0.707 Table 1: Dependence between fraction number and absorbance at 280nm. Week 7: Ion exchange chromatography A Q- Sepharose anion exchange column was used to separate a mixture of proteins. Before the actual experiment was started, the TA checked on the Gradifrac system. Then the recorder was set up with the required settings. The sample was injected into the sample loop, without any air bubbles. The following chromatogram was produced. Scheme 1. Chromatogram of the protein purification. Week 8: BCA Analysis 25 mL of BCA reagent was prepared in the ratio 50:1. For this purpose 24.5 ml of BCA reagent A was used and 0.5 mL of BCA reagent B. Then a standard curve was prepared as follows: Volume of the BSA Volume of H2O (dilutent) Final BSA concentration 100 µL (stock) 700 µL 250 µg/ mL (a) 400 µL (a) 400 µL 125 µg/ mL (b) 300 µL (b) 450 µL 50 µg/ mL (c) 400 µL (c) 400 µL 25 µg/ mL (d) 100 µL (d) 400 µL 5 µg/ mL (e) Table 2: Preparation of reagents for the standard curve. After standards and the unknowns were diluted and transferred to the microtubes, 2.0 mL of the reagent was added to each tube and mixed thoroughly with repeated pipetting. The mixture was then allowed to incubate at 37 °C for 30 minutes. Then all tubes were cooled to the room temperature and the absorbance at 562 nm was measured. Entry Absorbance A 0.533 B 0.298 C 0.176 D 0.109 Table 3: Measurement of absorption for different entries. Also, absorbance for each tube was measured: #1-0.116 (10%)- 10 µL of protein +90 µL H2O #2-0.187 (20%)- 20 µL of protein +80 µL H2O And the concentration of the protein was found to be = 337 µg/ mL=1.087 x10-5 mol/L Week 9: Activity assay. For this part, different substrate concentrations were tested to see their reaction rates. A reaction mixtures consisting of: Fold dilution Substrate (µL) Phosphate Buffer (µL) Protein (µL) 5x 200 800 25 10x 100 900 25 20x 50 950 25 40x 25 975 25 80x 12.5 987.5 25 were added to the appropriate cuvettes ( #1, #2, #3, #4, #5 )and then absorbance readings were recorded every 10 seconds. The produced results were used to perform the calculations. Calculations a) BCA Assay for mutant: graph and slope Scheme 2: BCA Assay for mutant: graph and slope b)Using Scheme 2 it is possible to determine protein concentration for IEC( GFC and crude exact given). The absorbance values for #1 and #2 determined in week 8 were used as “X” : (crude): #1-0.116 (10%)- 10 µL of protein +90 µL H2O: Concentration = 46.898?0.1162+510.18?0.116-34.716 = 25 µg/ mL (After GFC): #2-0.187 (20%)- 20 µL of protein +80 µL H2O Concentration = 46.898?0.1872+510.18?0.187-34.716 = 62 µg/ mL c) i)Enzyme Assay: Abs vs time for mutant (5 graphs per mutant): For crude: Scheme 3: Crude 5X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 4: Crude 10X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 5: Crude 20X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 6: Crude 40X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 7: Crude 80X (Fold dilution) (Time(X) Absorbance(Y)) After GFC: Scheme 8: After GFC 5X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 9: After GFC 10X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 10: After GFC 20X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 11: After GFC 40X (Fold dilution) (Time(X) Absorbance(Y)) Scheme 12: After GFC 80X (Fold dilution) (Time(X) Absorbance(Y)) c)ii) The reaction is followed by Michaelis-Menten kinetics, that is why its rate is given by: v0= where: v0 is the initial rate of the reaction; Vmax is the maximum rate of the reaction. If the amount of the enzyme is increased Vmax will also increase due to the following relationship: Vmax = kcat[enzyme] [S] – substrate concentration; Km- Michaelis constant, is used to characterise all enzymes; The reaction proceeds according to the following mechanism: In order to collect the required amount of data for the described equations the necessary amount of the substrate will be dissolved in a buffer solution. The reaction is started by the addition of the enzyme. The cuvette is introduced into a spectrometer to measure the absorbance at fixed time intervals. The reaction rate (?substrate/second) can be calculated in three steps: 1) Preparation of plots absorbance versus time for each substrate concentration (see above) 2) Calculation of the slope of the line (?absorbance/second). This step is done because v0 can be expressed as changes in absorbance per unit of time. 3) Divide the slope by the molar absorption of the substrate (?=5500 M-1 cm-1, b=1cm) If v0 is known for several substrate concentrations it is possible to determine Vmax and Km. The reaction is followed by Michaelis-Menten kinetics, therefore the dependence between [S] and v0 should have the form of a rectangular hyperbola. On the plot (v0/[S]), with the increase in the concentration of [S] the rate approaches Vmax. Also, Km is equal to substrate concentration at which v0=1/2 Vmax. Concequently, Vmax and Km can be determined using the plot. Using this method it is possible to determine Vmax and Km only approximately. To achieve more precise results a Lineweaver-Burke plot must be constructed (1/v0 versus 1/[S]). Taking into account the equation for Michaelis-Menten kinetics and taking its reciprocals the following equation will be produced: The equation is used to construct the Lineweaver-Burke plot. Example calculation from Scheme 3(Crude): 1) The values for the most linear region were taken: v0 = ?abs/?time = (1.072-0.076)/(300-10) = 0.0034/sec 2) Calculation of the rate using Beer-Lambert equation: A= ??c?l Therefore, c = A/(??l) where A = absorbance measured, ? = extinction coefficient, 5500 M-1 cm-1, c = concentration, l = the cuvette thickness, cm c = The result means that we 6.18 ? 10-7 M = 0.618 ? 10-6 M = 0.618 ?M which means 0.618 micromoles per liter or 0.618 nanomoles/mL generated in one second in the total volume of 1025?L = 1025 ? 10-6 L = 1.025 mL. The total amount of product formed in the whole volume is needed not the concentration. Taking into account this information the rate will be: 0.618 nanomoles/mL ? 1.025 mL = 0.633 nanomoles formed in one second or 0.633 nanomoles ? 60 sec = 38.007 nanomoles formed in one minute. Same calculations were done to all the presented above schemes: For crude: Fold dilution Substrate (µL) v0 (nanomoles formed in one minute) Protein (µL) 5x 200 38.00 25 10x 100 43.56 25 20x 50 4.67 25 40x 25 3.44 25 80x 12.5 1.74 25 Taking into account the starting concentration of the substrate 500 mg/ml the following table will be produced: [S] (mg/ml) V (nM/min) 1/[S] (ml/mg) 1/V (min/nM) 100 38.00 0.01 0.02600 50 43.56 0.02 0.00100 25 4.67 0.04 0.00054 12.5 3.44 0.08 0.00027 6.25 1.74 0.16 0.00014 This information allows to construct Michaelis-Menten and Lineweaver-Burke graphs: Scheme 13: Michaelis-Menten graph ([S]-X , V-Y) From the graph it can be clearly seen that Vmax = 46 nM/min and Km = 27 mg/ml (1/2Vmax = Km) Scheme 14: Lineweaver-Burke graph (1/[S]- X, 1/V0 – Y) The same logic can be applied to the sample that was produced after Example calculation from Scheme 8(After GFC): 1) The values for the most linear region were taken: v0 = ?abs/?time = (0.91-0.055)/(600-10) = 0.0014/sec 2) Calculation of the rate using Beer-Lambert equation: A= ??c?l Therefore, c = A/(??l) where A = absorbance measured, ? = extinction coefficient, 5500 M-1 cm-1, c = concentration, l = the cuvette thickness, cm c = The result means that we 2.63 ? 10-7 M = 0.263 ? 10-6 M = 0.263 ?M which means 0.263 micromoles per liter or 0.263 nanomoles/mL generated in one second in the total volume of 1025?L = 1025 ? 10-6 L = 1.025 mL. The total amount of product formed in the whole volume is needed not the concentration. Taking into account this information the rate will be: 0.263 nanomoles/mL ? 1.025 mL = 0.269 nanomoles formed in one second or 0.269 nanomoles ? 60 sec = 16.174 nanomoles formed in one minute. Same calculations were done to all the presented above schemes: After GFC: Fold dilution Substrate (µL) v0 (nanomoles formed in one minute) Protein (µL) 5x 200 16.17 25 10x 100 10.27 25 20x 50 7.87 25 40x 25 3.69 25 80x 12.5 6.95 25 Taking into account the starting concentration of the substrate 500 mg/ml the following table will be produced: [S] (mg/ml) V (nM/min) 1/[S] (ml/mg) 1/V (min/nM) 100 16.17 0.01 0.06184 50 10.27 0.02 0.09730 25 7.87 0.04 0.12700 12.5 3.69 0.08 0.27100 6.25 6.95 0.16 0.14388 This information allows to construct Michaelis-Menten and Lineweaver-Burke graphs Scheme 13: Michaelis-Menten graph ([S]-X , V-Y) From the graph it can be clearly seen that Vmax = 16 nM/min and Km = 25 mg/ml (1/2Vmax = Km) Scheme 14: Lineweaver-Burke graph (1/[S]- X, 1/V0 – Y) Conclusion: The modified enzyme possesses catalytic properties and can be successfully applied in the studied process. However, natural enzyme is still the most potent one. This fact highlights the importance of further research in the area. Works cited Mathonet, Pascale, Ulie Deherve, Patrice Soumillion, and Jacques Fastrez. “Active TEM-1 b-lactamase mutants with random peptides inserted in three contiguous surface loops.”, Protein Science 2006: 15:2323-2334. Print. Read More