Production, Purification and Characterization of OPH in Transformed E. coli w3110 ptrcHisB::oph

Kevin Kozikowski 1 December 2014

ChBE 168

Tufts University

I. ABSTRACT

Cell harvesting and protein purification by column chromatography are biotechnological techniques of utmost importance to the downstream processing of many bioprocesses. In this report, E. coli w3110 ptrcHisB::oph are induced and harvested in order to purify and characterize organophosphorus hydrolase, the most important enzyme in degrading toxic organophosphorus compounds. Bradford assay results indicate a high concentration of protein product in the third column volume, post IMAC purification. SDS-PAGE results indicate that purified product contains only one type of protein, whose size can be approximated to be 35 kDa. Activity assay results indicate that OPH produced in the lab is more active, but potentially less stable than OPH that is commercially available.

II. INTRODUCTION

Organophosphorus hydrolase (OPH) is a historically significant enzyme, playing a crucial role in the breakdown of toxic organophosphorus compounds often used in chemical warfare and pesticides.1 In this experiment, students will practice basic of downstream processing and protein characterization techniques and familiarize themselves with enzymatic catalysis, namely Michaelis-Menten kinetics.

The E. coli cells used in this experiment contain the ptrcHisB::oph plasmid and do not natively express OPH. The oph gene on the plasmid functions under the control of the lac promoter. Because the lac promoter is suppressed by a repressor protein unless galactose is present, oph is not actively transcribed. To promote the transcription and translation of the oph gene, IPTG is used as a galactose substitute to inhibit the repressor protein. As a result, IPTG induces E. coli to produce OPH.2

This experiment utilizes E. coli w3110 ptrcHisB::oph as an academic exercise, allowing students to practice downstream process techniques –cell harvest and purification. The use of centrifugation during harvest1 combined with Bugbuster3 disrupts the

cell membrane and separates aqueous proteins like OPH from membrane materials and cellular debris. Further purification is required, however, before the protein can be quantitatively characterized using SDS-PAGE. Chromatography is a widely used technique used to isolate particular proteins based on specific characteristics. An IMAC column uses metal ion chromatography to separate OPH from the mobile phase of the sample through the column.4 An effective column allows the protein of interest to elute off evenly, symmetrically centralized about a peak concentration. By collecting the sample in fractions based on column volumes and measuring their absorbance, the symmetry of the column can be determined by way of a pseudo-chromatogram. It is hypothesized that most protein should elute off of the column in the second and third column volumes, due to the dead volume of the column.

The Bradford Assay quantifies the amount of protein present in each sample by utilizing Coomassie Blue as a reagent whose color changes depending on the amount of protein present.5 Analyzing samples at 595 nm on a spectrophotometer allows for quantitative absorbance measurements to be made for standards of known concentration as well as samples of unknown

 Figure 1. IMAC pseudo-chromatogram, based on absorbance at 595 nm per one column volume sample fraction (0.5 mL).

concentrations. The absorbance of purified samples is then correlated to its total protein concentration by a prepared standard curve. This provides a measure of the productivity of the transformed E. coli.

The Bradford Assay, however, does not distinguish between proteins. Samples need to be further characterized in order to confirm that the quantity of protein measured in the collected samples is in fact OPH. SDS-PAGE is employed to provide insight into the size of the protein under analysis.6 Before utilizing this technique, samples are reduced in order to disrupt tertiary peptide structure so that it does not impact gel results. Then, SDS evenly coats the sample peptides with a negative charge so that samples move through the polyacrylamide at a rate proportional to its size when a voltage is applied across the gel. An NEB protein ladder is employed as a means of comparing unknown sample bands with bands of known mass.7 OPH is a homodimer, comprised to two 35 kDa subunits.8 Comparing unknown bands to the ladder bands, it is hypothesized that single bands at 35 kDa should be present in lanes containing sample fractions 2 and 3. Further, a comparison between purified and unpurified sample is conducted using SDS-PAGE. It is expected that unpurified sample will contain many bands of a range of masses, representing other proteins that are natively produced by the E. coli.

An enzymatic activity assay allows further characterization of the produced OPH. Unlike GFP, OPH is an enzymatic protein that works to hydrolyze toxic organophosphorus compounds into species that are nontoxic. The assay reaction can be generalized as follows:

The assay performed uses paraoxon (S) as a substrate, which when broken down yields yellow colored p-nitrophenol (P). By measuring absorbance at 400nm over time, the activity of OPH (E) can be calculated as a direct proportion to the change in absorbance over time. Activity is a measure of an enzymes ability to catalyze a particular reaction, therefore it is equal to the reaction velocity. The above reaction scheme yields the following expressions, which describe the kinetics of the catalyzed reaction.

By measuring the rate of product formation through the change in absorbance, the enzyme activity can be determined.

III. MATERIALS AND METHODS 50ml Flask Culture and Harvesting of E. coli Cells2

A 50 mL flask culture was prepared by diluting 1 mL of deep-freezer stock 1:10 with LB media. 50 uL of ampicillin stock was added to a bring the concentration to 50 ug/mL. Cultures were set to shake for 5.5 hr in 37°C. Then, cultures were induced by adding IPTG to a 0.4mM concentration from 100 mM IPTG stock. Cultures were incubated for 3 additional hours, then harvested by centrifugation at 5000 rpm for 10 minutes. E. coli Cell Disruption with Bugbuster Protocol3 Supernatant was disposed post centrifugation and the pellet was resuspended in 2.5 mL of Bugbuster and incubated for about 15 minutes on a rotator. The suspension was then centrifuged at 16000 g for 20 minutes. The supernatant was aspirated off and stored at -20˚C for further purification, and the pellet was discarded. IMAC Column Purification of Soluble Heterologous Proteins4 An IMAC column was prepared by snapping off the end and removing the stopper. A syringe was filled with deionized water and used to strip the ethanol used for storage of the column, working carefully to ensure that no air bubbles entered the column and that the column remained fully hydrated. Using the syringe, the column was then loaded with 5 mL of 50mM Tris buffer. Following, aqueous protein sample was loaded into the column and the column was rinsed with 5 mL of loading buffer. Following, 5 mL of elution buffer was loaded into the column and all flow-through was retained in 0.5 mL (1 column volume) fractions. Fractions were stored at -20˚C for further characterization. The column was then rinsed with 5 mL of DI and stored at 4˚C. Bradford Assay5

BSA standards at concentrations of 2000 ug/mL, 1500 ug/mL, 1000 ug/mL, 750 ug/mL, 500 ug/mL, 250 ug/mL, 125 ug/mL, 25 ug/mL and 0 ug/mL were prepared by diluting 2000 ug/mL BSA stock with DI. 20 uL of each standard and each sample to be tested were further diluted with 1 mL of Coomassie Blue reagent. The absorbance of each sample was measured at 595 nm using a UV-Vis spectrophotometer. Blank-corrected absorbances of the standards were used to create a linear standard curve, correlating absorbance with known protein concentration. The concentration of unknown sample fractions was determined based on

their absorbance by interpolation from the equation of the standard curve. SDS-PAGE Protocol6

Samples from fraction 1, 2, 3, 4, 5, 6, and 8, as well as crude cell extract (pre-IMAC purified sample) were prepared by mixing 10 uL of sample with 6 uL of Tris-SDS samle buffer and 2 uL of reducing agent. Each of the samples were heated at 85oC for 2 minutes. Precast gels were opened and assembled with the gel box. The box was filled with Tris-Glycine running buffer while checking the assembly for leaks. The gel was loaded with 18 uL of prepared samples according to the chart in Figure 4. NEB protein ladders were added to lanes 1 and 6. Gels were run at 150 V for about 90 minutes, and running buffer was saved. Gels were then removed from the plastic casing and rinsed with DI. Gels were then stained with Coomassie Blue reagent on a shaker for about 20 minutes, and allowed to destain in DI overnight.

OPH Assay9

100 uL of sample was combined with 100 uL of 250 mM CHES buffer, 200 uL of 1 mM paraoxon and 600 uL of DI. Immediately following this combination, Abs400 was measured every minute for a 20 min span.

IV. RESULTS

IMAC purified samples were collected in eight

fractions, each containing one column volume of sample.

Bradford Assay absorbance values were plotted against column volume in order to construct a pseudo-chromatogram (Figure 1). The asymmetry factor associated with the IMAC column could be approximated to be 1. OPH concentrations displayed in Table 1 were assigned to each fraction based on their absorbance value and a prepared standard curve. Assay results indicate that OPH concentration peaked in the third column volume at 66.78 ug/mL (Table 1). All other computed fraction values were negative. Generally, fractions declined in OPH concentration the further from fraction 3.

SDS-PAGE was employed to characterize purified samples (Figure 2). Protein markers occupy lanes 1 and 6, however the banding pattern is largely indistinct, with the bold bands at the top of the gel. A single band is present in lane 3, which contains purified sample from fraction 2, and could be roughly approximated to be 35 kDa. Lane 9, which contains crude cell extract, is an indistinct smear with a bold band corresponding to the same weight at that in lane 3. Visible bands are not present in lanes 2, 4, 5, 7 or 8, each of which contain non-peak sample fractions.

Enzymatic activity assay results indicate a nonlinear trend for OPH sample produced in the lab and a linear trend for the commercial OPH sample (Figure 3). The initial linear segment of the

experimental OPH sample is higher than that of the commercial sample at 0.0075 and 0.0064 respectively. These values were used to calculate enzyme activity of each sample according to the following equation:9

𝑣 = !"!"=

!"!"!"×1000× !"""  !"

!""  !"×𝐷

Table 1. Total protein concentration of sample fractions based on Bradford Assay.

Sample Fraction (Column Volumes, 0.5 mL)

Total Protein Concentration (ug/mL)

1 -45.44 2 -63.22 3 66.78 4 -39.67 5 -81.00 6 -73.22

 1 2 3 4 5 6 7 8 9 10

Ladd

er

Frac

tion

1

Frac

tion

2

Frac

tion

3

Frac

tion

4

Ladd

er

Frac

tion

5

Frac

tion

8

Unp

urifi

ed

Frac

tion

2

Figure 2. Unpurified and fractions of purified OPH SDS-PAGE results.  

where v is activity, !"!"

is the average slope of the absorbance plot, 𝜀 is molar extinction coefficient (17000M-1cm-1), l is length (1 cm), and D is dilution fold (1)

In this manner, the enzyme activities of experimental and commercial OPH were calculated to be 4.412E-3 μmol/min and 3.765E-3 μmol/min, respectively (see sample calculation in appendix).

V. DISCUSSION

The first phase of the experiment involved inducing OPH production in a flask culture of ptrcHisB::oph transformed E. coli cells with IPTG. IMAC allowed for both purification and fractionation of the sample. Utilization of the Bradford Assay allowed for the quantification of total protein concentration in each fraction of collected sample. A standard curve was prepared relating protein concentration to absorbance at 595 nm light. Fractions were assigned absorbance values in order to calculate their respective OPH concentrations based on a linear fit of the prepared standard curve.

With the exception of fraction 3, the reported protein concentrations from the Bradford assay for all fractions are negative. Because the results fall out of the absorption spectrum, they are not indicative of the true protein concentration. The sample level in the cuvette was not quite high enough for the particular UV-Vis instrument used, contributing to the flawed results. Additionally, the presence of impurities or a poor calibration may be factors contributing to the flawed

results. Despite obvious error, conclusions can still be drawn from the pseudo chromatogram, Figure 1. Looking at fractions relative to one another, the results indicate that OPH concentration peaked in the second column volume of sample. Fractions 2 and 4 are relatively balanced around the peak at fraction 3. The asymmetrical factor of the column is approximated to be As = 1, based on the number of column volumes in each direction from the peak where the absorbance is 10% of the peak absorbance. As the As target is 1, this value indicates that the column performed relatively well by controlling the passage of OPH through the column.

Purified OPH was characterized by SDS-PAGE. Fractions were compared in lanes 2, 3, 4, 5, 7 and 8; however, a band is only faintly visible in the lane containing sample fraction 2. This result contradicts results displayed in figure 3, which indicates that fraction 3 is the only fraction with a significant concentration of OPH. Due to the poor quality of the gel, it is difficult to draw any significant conclusions about the collected OPH sample. Only lane containing molecular weight marker shows very faint banding, making it very difficult to interpret the size of the purified sample. However, it is likely that the one dark band in lane 1 corresponds to the 27.0 kDa molecular weight marker because it falls relatively close to the sample band, with a reference value of 35 kDa.8 The smear that exists in Lane 9 indicates the presence of a range of different proteins in the crude sample, with a very dark band that aligns with the faint band that exists in lane three. The noticeable difference in banding patterns of the purified and unpurified samples indicates that IMAC effectively reduced host cell protein in the crude cell extract, however, it also significantly decreased the concentration of OPH.

The poor quality of the gel suggests that the gels were not allowed sufficient time to incubate with the stain. With a longer staining time, bands would be more effectively visualized on the gel, and would likely display a 35 kDa band in fraction 3. Further, the gel appears to be underdeveloped due to the clumping of bands near the top of the ladder-containing lanes. The dark 66.4 kDa band that is displayed in the literature from the NEB ladder manufacturer is not visible, suggesting that the gel was not run long enough to separate out heavier proteins. It is important that at least two distinct ladder band are visible to make any significant size measurements of unknown samples, which is not the case with the gel depicted above.

A kinetic enzymatic assay compared the activity of the OPH produced in the lab to a commercial OPH sample. A sample from fraction 3 was used to perform the assay because the Bradford assay indicated that it contained the highest concentration of OPH. Dr. Yi informed experimenters that the commercial sample

Figure 3. Enzymatic activity assay. Absorbance values over time for reactions catalyzed by commercial and experimentally produced OPH.

had a historically lower activity when compared to the experimental sample, however, on first glance, results indicate the opposite. Figure 3 indicates that the commercial sample catalyzes the hydrolysis much more quickly than the experimental sample, as is evident through the steeper slope. Additionally, the commercial sample behaves consistently throughout the observed runtime, showing a linear trend. These results are likely due to the extended wait time before running the assay. The OPH samples were not kept on ice while waiting for the preceding group to complete their assay, promoting the degradation of unstable experimental OPH. The commercial sample was not exposed to room temperature as long, and it is also likely engineered to be more thermally stable than the OPH produced in the lab. Considering these stability concerns, only the initial linear points from the experimental OPH curve were used to compute its activity. These points yielded a slightly higher slope than that of the commercial sample, indicating that if the sample had been kept on ice and was not subject to denaturation, it would have maintained a higher activity than the commercial sample throughout the assay. Activities of experimental and commercial OPH were calculated to be 4.412E-3 umol/min and 3.765E-3 umol/min, respectively (see sample calculation in appendix).

In conclusion, students were able to successfully induce a culture of transformed E. coli into producing OPH. Samples were purified and characterized quantitatively via SDS-PAGE in attempt to confirm that the collected sample protein matched the theoretical size of OPH. The Bradford Assay quantified the concentration of protein produced by the E. coli. OPH was confirmed to be active through an enzymatic activity assay, in which the activities of the experimental OPH and commercial OPH were compared. Through this experiment, students were able to practice downstream processing techniques and basic protein characterization via Bradford Total Protein Assay, SDS-PAGE, and an enzymatic assay.

VI. REFERENCES

1. McDaniel, Steven. Cloning and Sequencing of a Plasmid-Borne Gene (opd)Encoding a Phosphotriesterase. Texas A&M University, 1988.

2. Yi, Hyunmin. 50ml Flask Culture and Harvesting of E. coli BL21 Cells. Tufts University, 2014.

3. Yi, Hyunmin. E. coli Cell Disruption with Bugbuster Protocol. Tufts University, 2014.

4. Yi, Hyunmin. IMAC Column Purification of Soluble Heterologous Proteins. Tufts University, 2014.

5. INSTRUCTIONS: Coomassie (Bradford) Protein Assay Kit, Test Tube Procedures. Thermo-Scientific: 2.

6. Yi, Hyunmin. SDS-PAGE Protocol. Tufts University, 2014.

7. Protein Marker, Broad Range (2-212 kDa), New England BioLabs, 2012.

8. Mulbry, WW. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains. Pesticide Degradation Laboratory, U.S. Department of Agriculture, 1989.

9. Yi, Hyunmin. OPH Assay/Analysis. Tufts University, 2014.

VII. Appendix

Sample Calculation for Experimental OPH:

𝑣 =0.0075  𝐴𝑏𝑠

𝑚𝑖𝑛 ×𝑚𝑜𝑙 ∗ 𝑐𝑚17000  𝐿 ×

11𝑐𝑚×

10!𝜇𝑚𝑜𝑙𝑚𝑜𝑙

×𝐿

1000𝑚𝐿×1  𝑚𝐿  ×1000  𝜇𝐿100  𝜇𝐿 = 4.412×10!!

𝜇𝑚𝑜𝑙𝑚𝑖𝑛

 Figure A1. Standard curve prepared with BSA stock and DI. Absorbances were measured at 595 nm using a UV-Vis spectrophotometer, after preparing standards with Coomassie Blue reagent (as specified in Bradford Assay procedure).