Caleb Bhatnagar (31574125)
Kadandale, P. 4/12/2018
Cytochrome P450 Paper
Abstract
In this experiment, our goal was to identify a protein of interest from two E. coli that were masked. The main reasons this experiment was performed were to learn to identify a protein from bacterial strains and understand how it can be purified to increase specific activity. Additionally, understanding the purification steps from our experiment helps us understand the perspective of a scientist and think critically. Our addressed question in this experiment was which bacteria contained our Cytochrome P450BM3 and how the purification impacted enzyme characteristics, such as the specific activity. The important results from our experiment included absorbance, optical density, and specific activity measurements. We found that we were able to increase the amount of protein and its specific activity. Finally, we reflected on our experiment to identify errors, ways to improve, and potential experiments for the future.
Introduction
The purpose of this experiment was to learn how to transform and purify a plasmid from a bacteria cell. We used an E. coli that was previously engineered to contain our protein of interest. We used two different, unknown strains plasmid to complete each mini-prep. Various restriction enzymes were used to digest our protein and create maps to find which bacteria contains our gene of interest (the P450BM3 gene pT7BM3). The enzyme cytochrome P450BM3 is not naturally found in E. coli, the P450 protein in highly conserved in organisms ranging from bacteria to humans (1). The function of P450 varies in different organisms and its functions include breaking down drugs, vitamin and steroid production, and metabolizing cholesterol. The P450 has a heme prosthetic group containing a central iron atom and absorbs maximally at 450 nm in the carbon dioxide form (2). When placed in bacteria, this causes a reddish-brown color to appear. This group also is important for enzyme function as hydroxylases (or monooxygenases). Additionally, this enzyme enables a catalytic replacement of a substrate’s hydrogen with a hydroxyl group to occur. The hydroxylation reaction involves the transfer of electrons to an oxygen group, reducing it to a hydroxyl group and the second reduction to water.
Cytochrome P450BM3 was used specifically in this study as it is one the most studied and well known P450 proteins. This protein uses NADPH as its electron carrier to carry out hydroxylation of an oxygen group. The bacterial cytosolic flavocytochrome comes from Bacillus megaterium and can be produced in large quantities (3). It is 119 kDA and contains a heme-b-containing monooxygenase domain (BMO) that is fused to the reductase domain (BMR) with a FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) (4).
Results
In the first week of our experiment, our goal was to digest two unknown bacterial protein systems with various restriction enzymes. Following this experiment, we used various different techniques to identify which plasmid contained our gene of interested, the Cytochrome P450BM3 gene. An enzyme activity assay and Bradford assay were conducted to determine which protein had a higher activity and which sample had a higher concentration of protein (respectively). Our results were recorded in Figure 1. The enzyme activity and Bradford absorbance were higher for plasmid A compared to plasmid B. More specifically, the rate of reaction dropped faster for plasmid B in comparison to plasmid A and the R2 was slightly higher for the rate of reaction in plasmid B compared to plasmid A.
Figure 1a. This figure depicts an overview of our first enzyme activity results from our initial experiment with our plasmids. Also, in the figure are trendlines for each plasmid. The slope for plasmid A was -.0009 with an R2 value of 0.964. The slope for plasmid B was -.0093 with an R2 value of 0.982. 1b. This figure is an overview of our first Bradford assay results from our initial experiment with our plasmids. The absorbance of plasmid A was 0.176 and the absorbance of plasmid B was 0.171.
In order to determine to further confirm which bacteria contained our plasmid of interest, an absorbance graph was recorded for each sample (Figure 2). Absorbance 3 refers to the spectrum for plasmid A and Absorbance 4 refers to the spectrum for plasmid B. An important characteristic we looked for was a peak in the graph at 418 nm. This represents the presence of a heme group (4). Based off our data collection, we found Absorbance 4 (plasmid B) to have a peak – and thus could confirm that it contained our plasmid of interest.
Figure 2a. This figure depicts an overview of our absorbance scan results for plasmid A. Based on the result, this graph did not have a visible peak at 418 nm present. 2b. This figure depicts an overview of our absorbance scan results for plasmid B. Based on the result, this graph did have a visible peak at 418 nm present.
Following the first two weeks in our experiment, we wanted to purify our protein further. This was accomplished using salt fractionation techniques. After running our plasmid containing the P450 gene through different salt fractions of ammonium sulfate, we performed the same tests to see how our activity had changed. Our results are recoded in Figure 3. The change in absorbance was the largest for the 60-80% salt fraction and the smallest in the 0-40% fraction. Each rate of reaction was measured using the 340 nm wavelength. The Bradford assay was diluted to a 1:25 dilution. Based off the Bradford assay, the 0-40% fraction had the highest concentration of enzyme (1.9556 absorbance), followed by the 60-80% fraction (0.4502 absorbance), and lastly the 40-60% fraction (0.363 absorbance). However, our results were skewed as the 0-40% fraction absorbance did not fall within the range of our standard curve. We will explore possibilities for this error in the discussion section of the lab report. Following our salt fractionation, we placed our plasmid sample in tube for dialysis.
Figure 3a. This figure depicts an overview of our second enzyme activity results from our salt fractionation experiment with our plasmid B. Also included in this figure are trendlines for each reaction rate. The slope for 0-40% salt fraction was -.003 with an R2 value of 0.984. The slope for plasmid 40-60% salt fraction was -.0005 with an R2 value of 0.978. The slope for plasmid 60-80% salt fraction was -.021 with an R2 value of 0.978. 3b. This figure is an overview of our second Bradford assay results from our salt fractionation experiment with our plasmid B. The absorbance of plasmid B from our 0-40% fraction was 1.96. The absorbance of plasmid B from our 40-60% fraction was 0.362. The absorbance of plasmid B from our 60-80% fraction was 0.450.
In the last week of the experiment, we took our dialyzed plasmid sample and ran it through a gel filtration assay to further purify it (Figure 6). Following the filtration, several tests to check for enzyme activity. A final enzyme activity was run on the plasmid following the filtration and the slope recorded had a negative slope of 0.0006 (Figure 4a). The gel filtration, which separates proteins by size, was conducted on the ammonium sulfate fraction that had the highest activity (40-60%). Our Bradford assay was not diluted in the last week and the absorbance recorded was 0.0485, within our standard sample range (Figure 4b). To confirm that the sample we ran through the gel was still the P450 gene, another absorption spectrum was conducted to examine the presence of a heme group (Figure 5). Based off our data collection, we found the sample to have a peak at the 418 nm wavelength – and thus could confirm that it contained our plasmid of interest.
Figure 4a. This figure depicts an overview of our second enzyme activity results from our salt fractionation experiment with our plasmid B. Also included in this figure are trendlines for each reaction rate. The slope for the gel filtration assay was -0.0006 with an R2 value of 0.971. 4b. This figure is an overview of our third Bradford assay results from our gel filtration experiment with our plasmid B. The absorbance of plasmid B was 0.0485.
Figure 5. This figure depicts an overview of our absorbance scan results for plasmid B after gel filtration was conducted on our plasmid. Based on the result, this graph did have a visible peak at 418 nm present.
Figure 6a. This figure depicts a standard marker curve taken for the SDS-PAGE conducted in our experiment. In order to carry out the graph, the log of the molecular weight (Kd) was taken and graphed against a measured distance (cm). 6b. This figure depicts the SDS-PAGE gel after it was run and microwaved in our experiment. Bands are highlighted along each molecular weight. Our Gel filtrate had a P450 band at approximately the 119 Kd mark (green).
The gel SDS-PAGE was run through Denville Blue Protein Stain Protocol and microwaved before analysis (Figure 6b). Our results showed less bands appearing through each purification step. Overall, we identified a final P450 band at the approximately 119 Kd mark since it matches up with the green rung from the rainbow protein marker (5). Another calculation made from our results was a restriction map to calculate exactly how many base pairs our restriction enzymes had cut our P450 protein and how large the protein was. The results can be found below in Figure 7.
Cell Free Extract B 60-80% (NH4)2SO4 Salt Fraction Gel Filtration Extract
Total Extract Volume (mL) 10.1 9.0 2.4
O.D. from Bradford Assay 0.171 0.450 0.0485
Total mg Protein 127.8 136874.0 8169.0
Specific Activity (umoles NADPH oxidized per second per mg protein) 3.92 x 10-3 8.21 x 10-7 2.04 x 10-6
Total Activity (umoles NADPH oxidized per second) 0.502 0.0251 0.0167
Table 1. Table 1/2 hybrid. This figure contains results and mathematical calculations for various aspects of our experiment. Calculations were carried out using Cell Free Extract B since it contained our plasmid of interest. The specific activity initially decreased by salt fractionation but then increased after dialysis and gel filtration.
Figure 7a. This figure is an overview of the results from our gel electrophoresis with a standard marker for each restriction enzyme used on our Plasmid B. 7b. This figure is a construction of the plasmid map after cutting it with our restriction enzymes. The red is our P450 gene. 7c. This table is a summary of the base pairs lengths calculated for each of our digests (single and double). Total base pair length was also summed and included.
After completing all our experiments each week, our data was collected and calculated to check for purity. Through the various purification steps, several changes were recorded in our optical density, total protein, and specific activity (Table 1). The trends did not seem to move in one direction but rather increased through some techniques and decreased through others.
Discussion
The purpose of this experiment was to identify and purify a Cytochrome P450 gene from two unknown strands of E. coli. Through several weeks of experimentation, we were able to successfully complete our experimental questions. All tests we ran supported our expectations, with the exception of one sample in our Bradford assay for the salt fractionation assay. All of our results confirmed that plasmid B contained our protein of interest (Figure 1 and 2). Through our analysis, we were able to increase the total amount of protein from the start of our experiment. However, our specific activity did not increase compared to our first purification step (Table 1). This may be due to our protein of interest eluting in the salt fractionation column more in a larger amount than what we chose fir gel filtration. Additionally, the salt concentration can change how proteins interact with other solutes in the liquid environment (6). This may change the specific activity of our eluate.
Through the dialysis, we were able to increase the specific activity of our protein sample. This is indicative that the salt may have been inhibiting activity of protein from the salt fractionation purification (7). We know that our last step of gel filtration also helped to increase the purity of our protein based off our SDS-PAGE results (Figure 6b). Since less bands appeared through each step (the first two steps were smeared), we know that we were removing spectator molecules and narrowing down our sample to our protein of interest (8).
These results further our knowledge of P450 as we were able to understand how the enzyme is cut by various restriction enzymes. Additionally, we learned how a protein responds to different purification techniques. In the paper by Tsotsou et al., a mutant form of Cytochrome P450 was identified from E. coli and was purified using similar techniques as in our experiments. Their results were similar in the fact that they found their specific activity to increase and decrease through different purification steps. An absorption spectrum was also collected during their experiment to confirm the peak at 418 nm. There mutant protein had a different specific activity than the wildtype, which may be due to how the protein folded and interacted differently in solution. Another interesting aspect of their experiment was looking at the enzyme kinetics through Michaelis–Menten graphs to understand the steady state of the protein (9).
In our experiment, there were a few errors that we identified while conducting each experiment. The main error for our experiment was in our second Bradford assay; the value recorded for the 0-40% salt fraction was higher than our standard value. We had initially diluted our sample using a 1:25 dilution and increased it to a 1:100 dilution but still were not able to record a value within the standard range of the Bradford assay. For future experiments, we should conduct our Bradford assay carefully to make sure that our standards align with the measurement of our sample. Since our protein was highly concentrated, we should use more precise methods to measure salt when adding it to the fraction. The weighing scale we used did not have side walls to block air flow, which can interfere with our measurement. An interesting follow-up experiment from this would to understand and document the kinetics/interaction of the salt and protein.
References
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