Materials & Methods



Attached is sample beta-gal report that illustrates particularly well what I was looking for in the various sections. It is an actual student report that I modified slightly. If you had difficulties with any other sections in your report, it may be helpful to see how this report is different from yours. Please see me if you have further questions. JHD

An investigation of the kinetic parameters of purified E. coli b-galactosidase and its inhibition by IPTG

Abstract

β-galactosidase is an enzyme that catalyzes the breakdown of lactose into glucose and galactose, and it has been the subject of extensive previous studies. In this study, nickel-chelate affinity chromatography was used to purify a 6XHis-tagged form of β-galactosidase from a genetically engineered strain of E. coli to obtain purified enzyme for kinetic characterization. This procedure resulted in an 11% yield and a purification factor of 7. The success of the purification was confirmed by SDS-PAGE. The activity of the purified enzyme was measured using different concentrations of a synthetic substrate, ONPG; the calculated Km for this reaction was 0.075 mM, and the Vmax was 250,000 nmols ONP/min/mg protein. The galactose analog IPTG increased the Km for this reaction while having no effect on the Vmax, confirming prior reports that IPTG functions as competitive inhibitor of β-galactosidase. The calculated Ki for IPTG was 0.11mM. These results demonstrate the effectiveness of this one-step β-galactosidase purification strategy and enhance understanding of the catalytic mechanism of this enzyme.

Introduction

E. coli β-galactosidase is an enzyme that catalyzes the breakdown of lactose sugar into glucose and galactose (Karp, 2005). β-galactosidase is structurally similar to enzymes from various organisms, including humans (Jain et al., 1996) so understanding its function and mode of action is crucial to understanding the function of related enzymes.

The activity of β-galactosidase can be determined using ONPG, a synthetic substrate molecule that shares a similar two-ring structure with lactose and thus can be hydrolyzed by β-galactosidase to produce ONP (which can be measured spectrophotometrically) and galactose (Wallenfels, 1962). An ONPG analog, IPTG, can be used to determine the effects of an inhibitor on β-galactosidase activity. IPTG has one ring so it cannot be hydrolyzed by β-galactosidase, effectively inhibiting enzyme activity (Juers et al., 2003). As a result, IPTG most likely functions as a competitive inhibitor of β-galactosidase as described by Juers et al. (2003).

The lacZ gene regulates the production of β-galactosidase. In order to study the activity of this enzyme in vitro, E. coli cells were genetically modified with a plasmid that featured a constitutive promoter upstream of the lacZ gene for β-galactosidase. This promoter was regulated by IPTG, so over-production of β-galactosidase was induced with the addition of IPTG. In addition, the lacZ gene was modified to produce β-galactosidase with a 6XHis tail. This enabled enzyme purification by nickel-chelate affinity chromatography, because histidine forms stable coordination compounds with nickel ions (Porath et al., 1975). The purified enzyme was investigated using various spectrophotometric activity assays to determine its overall function and activity.

Materials & Methods

Induction of 6xHis-β-galactosidase

After removal of a 1-ml aliquot for gel analysis, a 200 ml culture of BL21 (pET-14b) E. coli cells in Luria broth (LB) media in log phase growth was induced with 1.0 mM IPTG for 1 hour at 37˚C. A 1-ml aliquot of the post-induction cells was saved for gel analysis. The two aliquots and the remaining induced cells were pelleted by centrifugation and stored at -70˚C.

Purification of 6xHis-β-galactosidase

The induced BL21 (pET-14b) E. coli cells were lysed and underwent protein extraction and purification using B-Per 6xHis Fusion Protein Purification Kit© (Pierce, Inc.). Information about the reagents in the B-Per 6xHis Fusion Protein Purification Kit© can be found at Pierce, Inc.’s web site (). For the experimental protocol see BISC 220 Lab Manual (Harris, 2007).

Total Protein Modified Bradford Dye Assay

1:5 dilutions of CE/PF in Z-buffer (60mM Na2PO4, 60mM NaH2PO4, 1mM MgSO4, 0.27% β-mercaptoethanol) were incubated with Protein Assay ReagentTM (Bio-Rad Laboratories, bio-) at room temperature for approx. 30 minutes. The absorbance of each assay solution at 595 nm was determined using a spectrophotometer with a range of 0.1-1A. A standard curve was generated with BSA diluted in Z-buffer to yield BSA concentrations from 0.1-0.8 mg/ml. Total protein concentrations of CE and PF were determined by comparison with the standard curve (see Appendix 1).

Specific Activity Assay

1:50 to 1:400 dilutions of CE, and 1:100 to 1:800 dilutions of PF in Z-buffer were prepared. Enzyme dilutions were added to Z-buffer in a 1:19 ratio of enzyme solution to buffer. 4 mg/ml ONPG was added to each solution in a 1:5 ratio of ONPG to solution. The reactions were incubated at 28˚C for 5 minutes and stopped with stop buffer (1M Na2CO3) in a 5:12 ratio of buffer to reaction mixture. The absorbance of each reaction mixture at 420 nm was determined using a spectrophotometer with a range of 0.1-2.5A. Specific activities of CE and PF were determined using the absorbance values of the PF and CE dilutions that were closest to 0.5 (see Appendix 2A). An aliquot of PF was removed for SDS-PAGE analysis. The remaining PF was mixed with glycerol in a 1:3 ratio of glycerol to PF and stored at -70˚C with the remaining CE.

Enzyme Kinetics Assay

A 3:800 dilution of the PF+glycerol solution was prepared in Z-buffer and added to reaction mixtures containing Z-buffer and ONPG, yielding final ONPG concentrations of 0.017 – 1.3 mg/ml. The reactions were incubated at 28˚C for 5 minutes, and stopped with stop buffer in a ratio of 5:12 buffer to reaction mixture. This protocol was repeated with the addition of IPTG (an ONPG inhibitor) to the reaction mixture at a final concentration of 0.025 mg/ml. The absorbance of each reaction mixture at 420 nm was determined using a spectrophotometer with a range of 0.1-1A. The enzyme’s specific activity at varying substrate concentrations was calculated.

SDS-PAGE Analysis

CE and PF samples were mixed 1:1 with Laemmli buffer (0.125M Trisma base, 4% SDS, 20% glycerol, 10% β-mercaptoethanol); pre- and post-induced E. coli cells were resuspended in 75 μl Laemmli buffer. The samples were boiled for 5 minutes. Five micrograms of the CE and PF samples and 10 µl of each whole-cell lysate, plus a commercial b-galactosidase standard and a molecular weight marker (Precision Plus ProteinTM Standards, Bio-Rad Laboratories) were separated on a 4-15% sodium dodecyl sulfate polyacrylamide gel run for approx. 45 minutes at 200V. Proteins were visualized with 30 min. Coomassie blue staining (10% acetic acid, 50% methanol, 0.25% Coomassie brilliant blue) followed by destaining (30% methanol, 10% acetic acid) overnight.

Results

To enable study of the kinetic properties of β-galactosidase, nickel-chelate affinity chromatography was used to purify a 6XHis-tagged version of the enzyme from a genetically engineered strain of E. coli that had been induced to produce elevated amounts of the protein. The success of the β-galactosidase induction and purification were determined using SDS-PAGE. Figure 1 illustrates that β-galactosidase production was increased with the addition of IPTG (the inducer in this system), because the band at ~100kD (the location of β-galactosidase, according to the control) is darker in the +IPTG lane than in the –IPTG lane. The purified fraction also contained a large amount of β-galactosidase in comparison with the crude extract which has a much smaller band at the ~100 kD mark.

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Figure 1: Successful induction and purification of 6XHis-b-galactosidase. A commercial β-galactosidase control, molecular weight standards, and samples of 6XHis-tagged β-galactosidase pre- and post-induction with IPTG, and before and after purification, underwent SDS-PAGE gel electrophoresis at 200V for 45 minutes. The proteins were visualized by Coomassie blue staining.

The amounts of total protein in the crude extract and purified fraction were determined using a modified Bradford assay, and the b-galactosidase activity present in these fractions was assessed in reactions with the synthetic substrate ONPG, which generates a product (ONP) that can be measured spectrophotometrically (see Appendices 1 and 2 for calculations). These data were used to calculate the total activity and specific activity for the CE and PF, as well as the percent yield and purification factor. These results, shown in Table 1, reveal that the purified fraction was reasonably pure since a high specific activity of β-galactosidase was obtained while the total activity decreased, resulting in a good percent yield and purification factor.

Table 1: Results of β-galactosidase enzyme purification. [pic]

To characterize the kinetic behavior of b-galactosidase and its response to the presumed inhibitor IPTG, the purified enzyme was reacted with varying concentrations of ONPG in the presence and absence of the inhibitor. The Michaelis-Menten plot of the activity data (Fig 2) illustrates that the reaction velocity eventually reached a plateau so the enzyme reached saturation point at relatively low substrate concentrations.

[pic]Figure 2: Michaelis-Menten plot of β-galactosidase activity in presence and absence of inhibitor. The specific activities of purified 6XHis-tagged β-galactosidase in varying substrate concentrations were calculated using a spectrophotometric assay. A range of ONPG concentrations were used, with and without the addition of 104.9 nmol/ml IPTG.

In order to determine the kinetic parameters associated with β-galactosidase, the activity data was used to generate a Lineweaver-Burk plot (Fig 3). The plots for the uninhibited and inhibited reactions had the same Vmax (same y-intercept), while the inhibited reaction had a larger Km (higher x-intercept). This pattern suggests that IPTG acts as a reversible inhibitor in the reaction of β-galactosidase with ONPG.

Figure 3: Lineweaver-Burk plot of β-galactosidase activity in presence and absence of inhibitor. The inverse substrate concentrations and specific activities from the Michaelis-Menten plot were used to generate this plot. Linear regressions were calculated using Excel.

The experimental values of Km, Ki, and Vmax were calculated from linear regression equations generated from the Lineweaver-Burk plot (see Appendix 3 for sample calculations). Table 2 compares these values with β-galactosidase kinetics data obtained from other studies. There are some discrepancies, but all values agree in terms of magnitude.

Table 2: Comparison of experimental and literature values for various β-galactosidase kinetic parameters.

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Discussion

The induction and purification of 6XHis-tagged β-galactosidase yielded a relatively pure fraction of enzyme that was subsequently assayed to obtain information about the kinetic behavior of β-galactosidase. Activity assays in the absence and presence of the galactose analog IPTG revealed that IPTG is a competitive inhibitor of the enzyme.

The results of SDS-PAGE (Fig 1) and the total and specific activity assays (Table 1) indicated the relative purity of the purified fraction. According to Porath et al. (1975), histidine and cysteine form stable complexes with nickel ions, so any proteins in the crude extract that had high concentrations of these amino acids would also be present in the eluted purified fraction after affinity chromatography. Although the purified fraction still had a few bands that were not seen in the commercial β-galactosidase control, these bands were fewer and less dark than the bands seen in the crude extract, indicating that the fraction had fewer contaminating proteins overall. The purification factor of 7.05 (Table 1) was fairly high, further indicating a high level of purity in comparison to the crude extract.

To confirm that the bands seen on the gel were fragments of β-galactosidase, a standard curve of mobility of protein bands of the molecular weight standards was generated (see Appendix 4). The size of the protein bands in the sample lanes was calculated, and the dark bands near 100 kD were determined to be 117.3 kD. β-galactosidase is a homotetramer and each monomer is 116.4 kD (Juers et al., 2003). Therefore, the observed fragments seem to be those of β-galactosidase monomers. To conclusively prove the identity of the fragments, Western blotting could have been performed with a β-galactosidase-specific antibody, but this size determination offered sufficient evidence that the enzyme was induced in E. coli cells, and that the purified fraction consisted mainly of β-galactosidase.

The effect of the inhibitor, IPTG, on the reaction of β-galactosidase with ONPG substrate was determined through specific activity assays. The Michaelis-Menten plot (Fig 2) of the activity data illustrated that ONPG did not cooperatively bind to the enzyme, and that the maximal velocity (Vmax ) could be reached at around 3000 nmols/ml when the enzyme was saturated with ONPG. The Michaelis constant (Km ) is the substrate concentration at one-half of Vmax and a small Km indicates that low substrate concentrations are required to reach one-half Vmax so the substrate has a high affinity for the enzyme (Karp, 2005). The Michaelis-Menten plot (Fig 2) thus illustrated that ONPG has a high affinity for β-galactosidase, because low ONPG concentrations were sufficient to reach Km. The Lineweaver-Burk plot (Fig 3) indicated that the inhibited reaction resulted in the same Vmax but larger Km than the uninhibited reaction. This pattern is consistent with competitive inhibition (Karp, 2005). IPTG is an ONPG analog (Roth & Huber, 1996) so it binds competitively to the active site of the β-galactosidase molecule and inhibits the enzyme. Thus, at low ONPG concentrations, the substrate was competing with IPTG for access to β-galactosidase active sites, resulting in an increased Km, since more ONPG molecules were needed to reach one-half of the maximal velocity.

The experimental values of Km, Km, and Vmax were the same magnitude as the values reported in the literature (Table 2) so reliable results were obtained. The literature value of Vmax was calculated at 20˚C (Wallenfels, 1962) but the experimental Vmax was determined at around 25˚C so the increase in temperature may have resulted in an increased reaction velocity. The experimental Km was less than the literature value (Juers et al., 2003), but this could be attributed to differences in the protocols that were used to study the enzyme kinetics. In addition, the functionality of the β-galactosidase assayed in this experiment may have been affected by the addition of the 6XHis tail. The extra amino acids may have caused conformational changes within the enzyme that facilitated substrate binding, leading to a lower Km. Overall, the experiment used a successful induction and purification protocol that resulted in the isolation of sufficient amounts of β-galactosidase for enzyme assays. The experimental kinetic parameters and inhibition data were consistent with the literature values, indicating that the activity assays provided reliable and accurate results.

The study of β-galactosidase is especially important because the enzyme is homologous to those found in several other organisms. In particular, the enzyme shares a homology with human β-glucuronidase that has already allowed researchers to determine the location of catalytically important amino acid residues in human β-glucuronidase by comparison to β-galactosidase structure (Jain et al., 1996). As the two enzymes share several common motifs and similar active sites, it is possible that human β-glucuronidase functions like β-galactosidase and the kinetics parameters of the two enzymes may be similar. Therefore, a better understanding of E. coli β-galactosidase can help researchers achieve a greater knowledge of structurally similar enzymes in other organisms and guide the study of the activity and inhibition of related enzymes.

References

BISC 220 Lab Manual: Cellular Physiology, 2007. Dept. of Biological Sciences, Wellesley College, Labs 1-4.

Jain, S., Drendel, W.B., Chen, Z., Mathews, F.S., Sly, W.S., Grubb, J.H. (1996) Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat Struct Mol Biol 3, 375-81.

Juers, D.H., Hakda, S., Matthews, B.W., Huber, R.E. (2003) Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase. Biochemistry 42, 13505-11.

Karp, G. (2005) Cell and Molecular Biology Concepts and Experiments, 4th edition. Hoboken: John Wiley & Sons, Inc.

Porath, J., Carlsson, J., Olsson, I., Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598-9

Roth, N.J., and Huber, R.E. (1996) GLU-416 of beta-galactosidase (Escherichia coli) is a MG2+ ligand and beta-galactosidase with substitutions for GLU-416 are inactivated, rather than activated by MG2+. Biochem Biophys Res Commun 219, 111-115.

Wallenfels, K. (1962) Beta-galactosidase (Crystalline). Methods in Enzymology 5, 617-663.

Appendices

Appendix 1 – Total protein assay

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Figure 4: BSA Standard Curve. A modified Bradford assay was used to generate a plot of absorbances at 595 nm at varying concentrations of BSA.

Total Protein sample calculation:

Absorbance of CE = 0.833

Using BSA linear regression equation (y = 0.833): x = 0.965

1:5 dilution, so final concentration = 0.965 × 5 = 4.83 mg/ml

Total amount of protein = concentration × volume of CE = 4.83 × 8.5 = 41.1 mg

Appendix 2 – Activity Calculations and Formulas

A: Specific Activity Sample Calculation

Absorbance of 0.1 ml 1:200 CE (4.83 mg/ml) dilution = 0.5617

Molar extinction coefficient of ONP = 4800 M-1 cm-1

Cuvette pathlength = 1.0 cm

[ONP] = 0.5617 / (4800 × 1.0) = 1.17 × 10-4 mols/L

Time of reaction = 5 minutes

[ONP] / time = 1.17 × 10-4 / 5 = 2.34 × 10-5 mols/L/min = 2.34 × 10-5 mmols/ml/min = 2.34 × 10-2 μmols/ml/min

Total volume of reaction tube = 3.4 ml

2.34 × 10-2 μmols/L/min × 3.4 = 0.08 μmols ONP/min/0.1ml

Total amount of protein = 4.83 mg/ml × 0.1 ml × 1/200 dilution = 0.00242 mg protein

Therefore, specific activity = (0.08 μmols/min/0.1ml) / (0.00242 mg protein) =

33.1 μmols ONP/min/mg protein

B: Total Activity

Total Activity = specific activity × total amount of protein

C: % yield

% yield = (total activity of CE / total activity of PF) × 100%

D: Purification Factor

Purification Factor = specific activity of PF / specific activity of CE

Appendix 3 – Calculating kinetic parameters

From Lineweaver-Burk linear regression equations (Fig 3):

y-intercept = 1/Vmax

x-intercept = -1/Km

Km+I = Km-I × (1 + [I] / Ki)

Appendix 4 – Determining molecular weights of protein bands

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Fig 5: Standard curve of mobility of protein bands. The migration distance of molecular weight standards on a 4-15% gradient polyacrylamide gel was measured using a millimeter ruler.

To determine molecular weight of unknown protein band, measure the migration distance of band and use linear regression equation to calculate x.

10x = molecular weight

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