Purification and Kinetic Phosphate Inhibition Analysis of Bacterial Alkaline Phosphatase

Transcription

1 Purification and Kinetic Phosphate Inhibition Analysis of Bacterial Alkaline Phosphatase Roshan Chikarmane CH 467 University of Oregon June 7, 26

2 Introduction Background Information. Alkaline Phosphatase (ALP) is a membrane-bound enzyme that dephosphorylates many phosphorylated substrates (), including lipids (2), nucleic acids (3), and proteins (4). Its primary biological role is to regulate Escherichia coli (E. coli) phosphate economy (5). As a laboratory tool, ALP is used as a protein label for enzyme-linked immunosorbent assays (ELISA) (6), is used to remove the 5 phosphate of nucleic acids prior to the addition of a radioactive phosphate group in DNA and RNA labeling (7), and is used as a staining target for the detection of pluripotent stem cells (8). Therefore, it is important that an efficacious method for generating ALP be devised so that it is readily available for use in such laboratory techniques. Purpose. The pet22b plasmid is an advantageous host for the gene for phoa since integrating it into the plasmid genome would make phoa inducible through activation of the T7 phage promoter with IPTG (9). Additionally, the pet22b plasmid can be extracted from and transferred to different strains of E. coli by transformation since it is distinct from the E. coli genome. In other words, pet22b can be efficiently extracted from bacteria, have the phoa gene integrated into its plasmid genome in vitro, and be efficiently transformed into a new bacterial colony in preparation for ALP biosynthesis. The goal of this study was to purify and characterize bacterial alkaline phosphatase (ALP) biosynthesized by a culture of transformed Escherichia coli (E. coli) strain BL2(DE3), which contained a recombinant pet22b-phoa vector. ALP biosynthesis was induced by IPTG, which disinhibited of the lac operator at the T7 phage promoter by binding to and sequestering the lac repressor and allowed the T7 polymerase to bind to the T7 promoter and transcribe phoa. After three hours of incubation, the cells were harvested and lysed with the detergent Triton X-

3 followed by two freeze-thaw cycles. The protein was extracted from the resulting lysate by DEA-Sepharose Ion Exchange Chromatography. Each of the purified protein fractions were subjected to an SDS-PAGE to qualitatively determine the varying sizes of the protein mixture in each fraction and an Immunoblot to qualitatively determine the trend of ALP abundance across the fractions. The trend of specific activities across the purified protein fractions was generated by consolidating data from an activity assay and a quantitative assay. Furthermore, the Km and Vmax of ALP was determined both with and without the presence of a phosphate inhibitor. 2

4 Results & Discussion The goal of this study was to purify and characterize bacterial alkaline phosphatase (ALP) biosynthesized by a culture of transformed Escherichia coli (E. coli) strain BL2(DE3), which contained a recombinant pet22b-phoa vector. The protein from a lysed culture of induced BL2(DE3) was purified by DEA-Sepharose Ion Exchange Chromatography. The resulting fractions were analyzed in the following ways. (a) Weight Profile of Protein Mixture in Each Fraction. Each of the purified protein fractions were subjected to an SDS-PAGE that was visualized by Coomassie Brilliant Blue R-25, a dye that binds nonspecifically with proteins (Figure ). The migration lengths of the Bio-Rad Figure. SDS-PAGE of purified protein fractions that have been stained by Coomassie Brilliant Blue R- 25. During purification Elution 3 mobile phase contained an NaCl concentration of 9 mm and each subsequent elution step was raised by 3 mm NaCl form the previous elution step, such that Elution 8 contained an NaCl concentration of 24 mm. Flow and Wash mobile phases contained no NaCl. Lysate was unpurified. Precision Plus Protein TM Dual Color Standard Ladder were plotted against the known masses designated to the bands and a best fit line with an R 2 value of.9787 was used to estimate the masses of proteins in the experimental bands (Figure 7 in Supplementary Information). Table summarizes the masses of proteins that were designated to the bands in each lane. Bacterial 3

5 alkaline phosphatase is known to be 43 kda. At least one band from the Lysate, Elution 3, Elution 4, Elution 5, Elution 6, and Table. Summary of Protein Masses at Each Band in Coomassie-Stained SDS-PAGE Lane Number of Bands Masses (kda) Lysate , 83.5, 45.5*, 39.*, 3.77 Wash 4.3 Flow No bands Elution , 99.4, 8.6, 53., 45.5* Elution , 99.4, 8.6, 53., 45.5* Elution , 99.4, 8.6, 53., 45.5*, 43.*, 35.7 Elution , 45.5* Elution , 6., 39.* Elution *Denotes masses within % of 43 kda, the known mass of ALP. Elution 7 lanes at least one band corresponds to a mass that was within % of the expected 43 kda. However, it is likely that the 45.5 kda bands correspond to ALP because it was the most reoccurring mass that came within % of 43 kda. These bands were the strongest, relative to other bands, in the Lysate, Elution 3, and Elution 4 lanes. (b) Qualitative ALP Concentration Variance Across Purified Fractions. The biosynthesized ALP was engineered to contain a His-Tag on its C-terminus, which was encoded on the pet22b vector immediately following the open reading frame of ALP. This His-tag was used as an affinity epitope for the primary antibody in the nitrocellulose Immunoblot gel (Figure 2). A secondary antibody bound to a fluorescent dye was bound to the primary antibody, thereby 4

6 Figure 2. Immunoblot on nitrocellulose of each purification fraction. Bands contained in all lanes except for the Bio- Rad Ladder lane denote ALP. labelling ALP with a fluorescent dye. The gel was visualized using a LI-COR Odyssey Fc system. The bands are the strongest in the Lysate, Elution 3, and Elution 4 lanes which was consistent with the ALP bands seen in the Coomassie gel. ALP bands in the Elution 5, Elution 6, Elution 7, and Elution 8 lanes are strong, but to a lesser degree. The Wash lane shown no ALP content and the Flow lane shows only slight ALP content. (c) ALP Activity Across Purified Fractions. ALP is known to catalyze the hydrolysis of phosphate monoesters. Therefore, ALP catalyzes the dephosphorylation of colorless p- nitrophenyl phosphate (PNPP) to form yellow p-nitrophenol (PNP). The colorimetric assay that was used tracked the rate of appearance of yellow color using UV-vis spectrophotometry to infer the catalytic activity of ALP in a given fraction. Figure 3 shows the absorption spectrum of a standard 5 μm solution of PNP, which was used to determine the extinction coefficient (ε) of PNP and the maximum wavelength (λmax). The λmax was determined to be 5

7 Absorbance Wavelength (nm) Figure 3. Absorbance Spectrum for PNP. at a wavelength of 4 nm with an absorbance of.62 at that wavelength. The extinction coefficient at 4 nm (ε4) was calculated using the Beer-Lambert Law as follows, where A4 denotes the absorbance at a wavelength of 4 nm: ε 4 = A 4 [PNP] (Path length) =.62 (.5 M) ( cm) = 24 M cm The A4 was recorded every. minutes for minutes for each fraction to determine the change in A4 over time (da4/dt) in min -. The change in PNP concentration (d[pnp]/dt) over time was calculated in terms of μmol/min as follows: d[pnp] dt in μm min = (da 4 dt ) ε 4 ( cm) ( 6 μmol ) mol 6

8 The d[pnp]/dt was taken to be the activity of ALP. Table 2 summarizes the activities of each fraction. Figure 8 in Supplementary Information shows Activity (μm PNP/min) versus NaCl Concentration (mm) in the elution mobile phase during purification. Table 2. Activity of Purification Fractions Fraction Activity (μm PNP/min) Lysate.5575 Flow.5423 Wash.3642 Elution.2446 Elution Elution Elution Elution Elution Elution Elution Elution Elution.793 Figure 9 in the Supplemental Information section shows the A4 versus time (min) plots for each purification fraction. (d) Protein Concentration Across Purified Fractions. A colorimetric assay known as the Bradford Assay was used to determine the protein concentration of each purification fraction. The absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-25 shifts from 465 nm to 595 nm when the dye binds to proteins. The A595 values of standard BSA solutions incubated in Coomassie was recorded and used to calculate the extinction coefficient of Coomassie at 595 nm (ε595). BSA was used because its size is similar to the size of alkaline phosphatase. Figure 4 shows a plot of the A595 versus BSA mass (mg). 7

9 BSA Mass (mg) (a).25.2 Figure 4. (a) BSA Mass versus A595 (b) Yields this equation (R 2 =.997) A 595 (b) Protein Mass in mg = (.472 A 595 ) +.3 BSA mass was calculated by multiplying the concentrations of the various BSA dilutions (mg/ml) by the volume of solution used in each sample (ml). Figure in the Supplemental Information section shows the absorption spectra of BSA standards at various concentrations. The resulting equation was used to estimate the mass of protein in each fraction based on the recorded A595 value. Table 3 summarizes the protein content of each purification fraction. Figure in the Supplemental Information section shows the absorbance spectra of the various purification fractions. 8

10 Table 2. Protein Content of Purification Fractions Fraction Protein Mass (mg) Lysate.249 Flow.38 Wash.8 Elution.275 Elution Elution 3.2 Elution Elution 5.22 Elution 6.27 Elution Elution Elution 9.23 Elution.58 (e) Specific Activity Across Purified Fractions. The activity (μm PNP min - ) was divided by the protein content (mg protein) for each purified fraction to yield the specific activity (μm PNP min - mg protein - ). Figure 5 shows a plot of Specific Activity (μm PNP min - mg protein - ) for each purification fraction. Table 3 gives the values of Specific Activity (for each purification fraction. 9

11 Specific Activity (μmol PNP/min-mg protein) Purification Fraction Figure 5. Specific Activity for each purification fraction. Table 3. Specific Activity of Purification Fractions Fraction Specific Activity* Lysate 2.24 Flow.392 Wash.338 Elution.89 Elution 2.99 Elution Elution 4 8. Elution 5.7 Elution 6.9 Elution Elution 8.26 Elution 9.46 Elution.3 *μm PNP min - mg protein -

12 (f) Kinetic Analysis of Bacterial ALP without Phosphate Inhibitor. ALP catalyzes the dephosphorylation of colorless p-nitrophenyl phosphate (PNPP) to form yellow p- nitrophenol (PNP). The colorimetric assay that was used tracked the rate of appearance of yellow color using UV-vis spectrophotometry to infer the catalytic activity of ALP in a given fraction. Table 4 shows the determination of the extinction coefficient of PNP at 4 nm (ε4) at various concentrations of PNP. The average ε4 was taken. Figure 2 in the Supplementary Information shows the absorption spectrum of a standard solutions of PNP, which were used to determine the extinction coefficient of PNP at 4 nm (ε4). Table 4. Determination of ε4 of PNP [PNP] (μm) [PNP] (M) Path Length (cm) A4 Extinction Coeff. Ε (M - cm - ) 5 5.E E E *Undiluted stock solution; εaverage = M - cm -. Table 5 summarizes the results from the Kinetic Assay. Initial ALP concentration was calculated as follows: Volume of Diluted Soln. Added to Rxn. [ALP] = [ALP stock] (Dilution Factor) ( ) Total Volume of Rxn. = (2 mg ) ( μl mg ) ( ) =.2 = 2 μg ml μl ml ml for all reaction mixtures.

13 Table 5. Summary of Results from Kinetic Assay Trial # [PNPP] (μm) d[pnp]/dt (μm/min) /[PNPP] (μm - ) /d[pnp]/dt (min/μm) μg/ml alkaline phosphatase (ALP) for each sample; Vtotal of all reaction mixtures is ml; Vmax = 6.4 μm/min; Km = μm. Initial PNPP concentrations were calculated as follows: [PNPP] = [PNPP stock] (Dilution Factor) (Volume of Diluted Soln. Added to Rxn. ) (46 g mol ) = ( mg ) ( 9 μl g ) ( ) (46 ) = ml μl mol.95 5 M = 9.5 μm for Trial # = ( mg ) ( 9 μl g ) ( ) (46 ) = ml 8 μl mol M = 22.4 μm for Trial #2 = ( mg ) ( 9 μl g ) ( ) (46 ) = ml 5 μl mol M = 39. μm for Trial #3 = ( mg ) ( 9 μl g ) ( ) (46 ) = ml 2 μl mol M = 97.5 μm for Trial #4 = ( mg ) ( 6 μl g ) ( ) (46 ) = ml μl mol 3. 5 M = 3. μm for Trial #5 = ( mg ) ( 9 μl g ) ( ) (46 ) = ml μl mol M = 95. μm for Trial #6 Initial Activity values were calculated in terms of μm/min as follows: d[pnp] dt in μm min = (da 4 dt ) ε average ( cm) ( 6 μmol ) mol 2

14 The kinetics constants Vmax and Km were calculated as follows: V max = (Y intercept of K m = V max (slope of d[pnp] dt d[pnp] dt vs. ) = [PNPP] = 6.4 μm/min.56 min/μm vs. ) = (6.4 μm ).583 min = μm [PNPP] min (g) Kinetic Analysis of Bacterial ALP with Phosphate Inhibitor. Table 6 summarizes the results from the Kinetic Assay. Table 6. Summary of Results from Inorganic Phosphate Inhibitor Analysis Trial # [PNPP] (μm) d[pnp]/dt (μm/min) /[PNPP] (μm - ) /d[pnp]/dt (min/μm) μg/ml alkaline phosphatase (ALP) for each sample;. mm Inorganic Phosphate for each sample; Vtotal of all reaction mixtures is ml; Vmax = 5.22 μm/min; Km = μm. Initial ALP concentration was calculated as follows: Volume of Diluted Soln. Added to Rxn. [ALP] = [ALP stock] (Dilution Factor) ( ) Total Volume of Rxn. = (2 mg ) ( μl mg ) ( ) =.2 = 2 μg ml μl ml ml Initial Phosphate concentration was calculated as follows: for all reaction mixtures. Volume of Diluted Soln. Added to Rxn. [P i ] = [P i stock] (Dilution Factor) ( ) Total Volume of Rxn. = ( mm) ( μl ) ( ) =. mm for all reaction mixtures. μl 3

15 Initial PNPP concentration was calculated as follows: [PNPP] = [PNPP stock] (Dilution Factor) (Volume of Diluted Soln. Added to Rxn. ) (46 g mol ) = ( mg ) ( 8 μl g ) ( ) (46 ) = ml μl mol.73 5 M = 7.3 μm for Trial # = ( mg ) ( 8 μl g ) ( ) (46 ) = ml 2 μl mol M = 86.7 μm for Trial #2 = ( mg ) ( 8 μl g ) ( ) (46 ) = ml μl mol M = 73.4 μm for Trial #3 Initial Activity values were calculated in terms of μm/min as follows: d[pnp] dt in μm min = (da 4 dt ) ε average ( cm) ( 6 μmol ) mol The kinetics constants Vmax and Km were calculated as follows: V max = (Y intercept of K m = V max (slope of d[pnp] dt d[pnp] dt vs. ) = [PNPP] = 5.22 μm/min.96 min/μm vs. ) = (5.22 μm ) min = μm [PNPP] min Figure 6 shows a Lineweaver-Burk plot of ALP without phosphate inhibitor (black) and with phosphate inhibitor (orange). 4

16 /V (min/μm).2 Lineweaver-Burk Plot /[PNPP] (μm - ) Figure 6. Lineweaver-Burk plot of ALP without phosphate inhibitor (black) and with phosphate inhibitor (orange). The equation (R 2 =.9999) for the uninhibited plot (black) is as follows: in min v μm = (3.346 in μm [PNPP] ) +.96 The equation (R 2 =.999) for the inhibited plot (orange) is as follows: in min v μm = (.583 in μm [PNPP] ) +.56 When inhibited the ALP Vmax decreases by 8.6 % and Km decreases by 6.2%, which indicates either mixed inhibition or noncompetitive inhibition. However, phosphate is known to bind to the catalytic site so it is more likely mixed inhibition since noncompetitive inhibitors do not bind to the active site. 5

17 Activity (μmol PNP/min) Mass (kda) Supplemental Information (a) (b) Migration Distance (cm) Mass (kda) = 83.6 (Migration Distance in cm).379 Figure 7. (a) Mass (kda) versus Migration distance (cm) of Bio-Rad Precision Plus Protein TM Dual Color Standard Ladder in Coomassie-stained SDS-PAGE. (b) Equation of best fit line (R 2 =.9787) that was used to infer the masses of experimental bands at their respective migration distances [NaCl] in Elution Buffer (mm) Figure 8. Activity versus [NaCl] in elution mobile phase during purification. 6

18 Absorbance A 4 Lysate Time (minutes) Figure 9. A4 versus time (min) for each purification fraction. Flow Wash Elution Elution 2 Elution 3 Elution 4 Elution 5 Elution 6 Elution 7 Elution 8 Elution 9 Elution [BSA] = 2 μg/ml [BSA] = 4 μg/ml [BSA] = 8 μg/ml [BSA] = 2 μg/ml [BSA] = 6 μg/ml [BSA] = 2 μg/ml Wavelength (nm) Figure. Absorption spectra of BSA standards at various concentrations. 7

19 Absorbance Absorbance [BSA] = 2 μg/ml [BSA] = 4 μg/ml [BSA] = 8 μg/ml [BSA] = 2 μg/ml [BSA] = 6 μg/ml [BSA] = 2 μg/ml Wavelength (nm) Figure. The absorbance spectra of various purification fractions {PNP] = 5 um [PNP] = 5 um [PNP] = 25 um Wavelength (nm) Figure 2. The absorption spectrum of standard solutions of PNP. 8

20 References. Bessey, Otto A., O. H. Lowky, and Mary Jane Brock. "A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum."journal of Biological Chemistry 64 (946): Snyder, Fred, and Merle L. Blank. "Specificities of alkaline and acid phosphatases in the dephosphorylation of phospholipids." Biochemistry 9.25 (97): Richardson, Charles C. "Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli." Proceedings of the National Academy of Sciences of the United States of America 54. (965): Swarup, G., S. Cohen, and D. L. Garbers. "Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases." Journal of Biological Chemistry (98): Horiuchi, T., S. Horiuchi, and D. Mizuno. "A possible negative feedback phenomenon controlling formation of alkaline phosphomonoesterase in Escherichia coli." (959): Halbert, Seymour P., and Milton Anken. "Detection of hepatitis B surface antigen (HBS Ag) with use of alkaline phosphatase-labeled antibody to HBS Ag." Journal of infectious diseases 36.Supplement 2 (977): S38-S Chaconas, George, and Johan H. van de Sande. "[] 5-32 P labeling of RNA and DNA restriction fragments." Methods in enzymology 65 (98): Kerr, Candace L., Hill, Christine M., Blumenthal, Paul D., Gearhart, John D. "Expression of pluripotent stem cell markers in the human fetal testis." Stem Cells 26.2 (28): &lin=f&keep=&srchmode=&unlock 9