Expression and Purification of CelB2 from E. Coli in Determining its Functionality in Cellulosic Biofuel Production

Nicholas Amado

Abstract

CelB2 is an immensely important enzyme responsible for the hydrolysis of cellulose. This functionality provides practical applications such as in cellulosic biofuel production. This process allows cellulose to be converted into glucose which can be subsequently fermented into ethanol and other fuels. The problem, however, is that cellulase enzymes such as CelB2 are catalytically slow and less active at higher temperatures. This severely hinders its efficiency as a catalytic converter of fuel and therefore CelB2 needs further study. The purpose of this experiment is to understand how to best express and purify CelB2 and subsequently study its functionality. CelB2 is expressed as a His 6MBPCelB2 fusion as this allows for better expression in E. Coli and subsequent purification. Purification of this fusion by amylose affinity chromatography was successful as shown by SDS-PAGE and absorbance at 280 nm. Factor Xa was used to cleave this fusion and its efficiency was analyzed by SDS-PAGE. Further purification by DEAE chromatography eliminated almost all residual proteins, but also removed most of CelB2 as shown qualitatively in SDS-PAGE and quantitatively in the purification table.

Introduction

Our dependence on fossil fuels is a serious issue that has galvanized researchers to develop plant derived fuel products. The current method for producing ethanol from corn and other plants is inefficient. Of emerging interest is the potential cellulose has as a biofuel. Cellulose is believed to be the best solution to this problem, using low-cost biomass for production. Cellulose undergoes cleavage by cellulase enzymes present in many microorganisms during metabolism. This process allows cellulose to be converted into glucose and glucose can be fermented into ethanol and other fuels. The only problem is that cellulosic biofuel production is not fit for an industrial scale. Cellulase enzymes are typically catalytically slow and lose activity and stability at high temperatures. Therefore, to study CelB2 we need to express CelB2 as His6MBPCelB2 fusion and purify using amylose affinity chromatography, Factor Xa digest, and DEAE chromatography. These methods should allow proper expression and purification of CelB2 for further study of its properties.

Materials and Methods

Expression. We streaked the Hi96MNPCelB2 strain of E. Coli on a LB agar plate. The agar plate consisted of a solution of 1 L water, 10g NaCl, 15g agar, 10g tryptone, and 5g yeast extract. This recipe makes about 50 plates of 100 mm diameter. The final result of the streaking is pictured in Figure 1. We then picked a colony from the plate with a pipette tip, swirling it in the starter culture. This was done 16-18 hours before induction. For inoculation, we used 1:100 dilution of starter culture into the prepared Luria Broth (LB contains 500 mL water, 5g tryptone, 2.5g yeast extract, and 5g NaCl) in the expression flask. The started culture contained 5 microliters of ampicillin. The expression media consisted of 500 mL LB, .5 mL (1:1000 dilution) ampicillin, and 2 mL of 50% (.2%) glucose solution. Time until OD 600nm = 0.6 was recorded at 2 hours and 10 minutes. The exact value of absorbance was recorded at .59 at this time. Induction was ready to be

started at this point and 1 mL of IPTG was added; solution then grown for 3 hours. Cells were then harvested by centrifugation at 4000 rpm for 20 minutes. Finally, the pellet from expression is stored at -20 degrees Celsius.

Plasmid Purification. Plasmid pMALCelB2 was purified from BL21(DE3) starter culture according to Qiagen Miniprep kit protocol. All steps were carried out at room temperature. Before beginning the protocol, 1 mL LB was added to the bacterial cell pellets in a 1.5 mL microfuge tube and centrifuged for 5 minutes. The LB supernatant was discarded and this process was repeated for a total of 5 times. Bacterial cell pellets were then resuspended in 250 µl Buffer P1 (4 °C on ice) and vortexed. 250 µl Buffer P2 was added to the microfuge tube after ensuring no clumps remaining in the suspended pellet and thorough mixing was ensured through inversion 4-6 times. Next, 350 µl Buffer was added and mixed immediately by inverting 4- times until the solution became cloudy. The microfuge tube was then centrifuged at 13,000 rpm for 10 minutes in a microcentrifuge. Supernatant was transferred to QIAprep spin column and centrifuged for 30-60 seconds; flow-through subsequently discarded. QIAprep spin column was then washed with .5 mL Buffer PB in order to remove trace nuclease activity and centrifuged again for 30-60 seconds; flow-through subsequently discarded. Spin column was washed again by adding .75 mL Buffer PE and centrifuging for 30-60 seconds; flow-through subsequently discarded. The QIAprep spin column was then centrifuged at full speed for 1 minute to remove any remaining wash buffer. DNA was eluted from the QIAprep column with 40 L dH2O and let stand for 1 minute, and then centrifuged for 1 minute.

Plasmid Restriction Analysis. Plasmid pMALc4CelB2 restriction was carried out with enzymes EcoR I and Hind III. 5 µl EcoR I was added to a solution of 10 µl plasmid DNA, 5 µl 1X Buffer, and 20 µl dH 2O and incubated at 37 °C. After approximately 1 hour of incubation, a 10 µl aliquot was taken for the agarose gel. This aliquot contains the single cut DNA. 5 µl Hind III was added to the incubation and after another hour, a 10 µl aliquot was taken for the agarose gel. This aliquot contains the double cut DNA. Lastly, 1% agarose gel was run containing the Bio-Rad cat #170-8354 ladder, uncut plasmid, and the single and double cut plasmids that was prepared during restriction.

Cell lysis was carried out by re-suspending 250 mL of the expression pellet in 5 mL of 20 mM Tris Buffer, 0.2 M NaCl, and 1 mM EDTA, making sure to fully dissolve the pellet. Then, 0.25 mg lysozyme for every mL of re-suspended pellet solution (2.5 mg were added for 10 mL of pellet solution). This solution was stirred on a shaker for 20 minutes. The solution was then sonicated on ice in 10 sec pulses for a total of 5 minutes. After 20 minutes, 200 µL protamine sulfate was added to the expression pellet suspension and centrifuged for 30 minutes at 10,000 rpm. Centrifugation was performed a second time for 30 minutes at 10,000 rpm to ensure clear separation of supernatant and pellet. A 20 µL aliquot of the supernatant was taken and placed into a 1.0 mL centrifuge tube to run on SDS-PAGE, and a 100 µL aliquot was also taken for subsequent activity measurement. The rest of the supernatant was transferred into a 50 mL conical tube and stored at -20 C.

Amylose Affinity Chromatography. CelB2 purification by amylose affinity chromatography was carried out. The amylose resin was prepared and loaded via Batch method. To prepare the resin, 10 mL of 20 mM Tris HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA was taken and added to 3 mL of amylose resin in 20% ethanol in a centrifuge tube. This solution was mixed and then centrifuged at 500 rpm for 3 minutes. The remaining buffer was decanted after centrifugation and another 10 mL of 20 mM Tris HCl (pH 7.4), 0.2 M NaCl, 1 mM EDTA was added, centrifuging again at 500 rpm for 3 minutes. Once completed, the buffer was decanted and the supernatant stored from the cell lysis experiment was added to the resin and incubated on a shaker at 4C for 1 hour. After incubation, the column was prepared by filling half full with wash buffer (20 mM Tris HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA) and tapping the sides to dislodge any air bubbles. The resin was swirled to achieve homogeneous suspension and then added to the column to achieve the desired resin-bed height (about 2 cm); flow-through was collected at this step. Once the resin bed settled, the column was washed with 25 mL wash buffer and collected. After this, the column was ready for elution. The column was washed with elution buffer (20 mM Tris HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA, 10 mM maltose) and collected in 7 fractions, 1.0 mL each. The maltose in

the elution buffer is responsible for binding to the MBP-CelB2 in the resin and separating it from the amylose. After collecting each fraction, 20 µL aliquots were collected from these fractions in order to run SDS-PAGE. All solutions and fractions collected were stored at 4C. The remaining resin in the column bed was washed with 3 column volumes of dH2O, followed by 3 column volumes of 0.1% SDS, followed by 6 more column volumes of water, and then resuspended in 20% ethanol and saved.

Factor Xa Digest. Affinity chromatography elutions 1, 2, 3, 4 and 5 were combined with an estimated total concentration of .5 mg/mL. 10 L of 1 mg/mL factor Xa stock solution was added to this protein solution and mixed by gently pipetting up and down. This solution was allowed to “digest” by incubated overnight at 23 C.

Buffer Exchange. For the buffer exchange, the Microsep Advance 3K centrifugal device was used. The MWCO 3K was chosen as this allows maximum retention for the protein of interest. A pre-rinse was carried out first to remove any trace extractables. The pre-rinse was carried out with dH20 by pipetting the dH20 into the filter device and spinning in the centrifuge for 10 min at 4000 rpm; excess water was poured out. Next, the protein solution was pipetted into the filter device and centrifuged for 30 min at 4000 rpm. In order to reduce the volume; excess solution poured out. To re-concentrate and reconstitute with exchange buffer, piperazine was pipetted into the filter device and centrifuged for 30 min at 4000 rpm. This step was repeated 3 times with the last two steps at 20 minutes instead of 30; excess solution not trapped in the filter was poured out each time.

Ion Exchange Chromatography. A weak anion exchanger such as DEAE (diethylaminoethyl) sepharose was prepared as the resin. About 16 mL of DEAE-sepharose and 20 mL of 20 mM piperazine buffer, pH 6.0, was combined and pipetted into the column to give a bed height of 10 cm. The piperazine-HCl buffer, pH 6.0, is responsible for equilibration, associated all of the charged groups in the stationary phase with ions of the opposite charge. That said, the column was washed with 5 column volumes of 20 mM piperazine buffer, pH 6.0 and once equilibrated, the protein sample was loaded onto the column; flow-through was collected in 2.0 mL fractions. At this point, elution of the proteins in the column was ready. A gradient of 0 mM to 700 mM NaCl piperazine buffer, pH 6.0, was made by separating the two beakers containing 25 mL of each buffer (0 mM and 700 mM NaCl) with an elastic tubing bridge. The affinity the proteins have towards the ion-exchange resin decrease as ionic strength increases, and therefore, as our NaCl concentration gradually increases, the salt will compete for binding in the stationary phase and elute the proteins. The different proteins will separate because the amount of NaCl needed to compete differs with the charge of each protein. These fractions were collected in about 1 mL volumes until the salt bridge was nearly done; this resulted in 39 fractions which were subjected to absorbance at 280 nm readings.

End Point Activity Assay. An activity assay was carried out to determine the activity of CelB2 is in our fractions after DEAE ion exchange. To do this, we used the substrate para-nitrophenyl -D-cellobioside (pNPC). CelB2 breaks down pNPC to yield pNP and thus the activity of CelB2 can be detected by measuring the absorbance after the reaction. A 100 L aliquot of each fraction was mixed with 100 L substrate (1 mg/mL pNPC stock) and incubated at 50 C for 30 min. During incubation, a 12 % SDS-PAGE was set up. The gel was run at 85 V for 15 min and then 135 V until complete. The gel is reported in the results as Figure 5. The reaction was stopped by adding .1M NaOH (2M NaOH stock) which also develops yellow color in the presence of pNP. A 200 L aliquot of each fraction was taken and 800 L H2O was added to this to reach a total volume of 1 mL. The absorbance for each fraction was measured at 405 nm and reported in Table 2. Water was used as the blank and the control contained 100 L phosphate buffer and 100 L pNPC substrate.

Results

Figure 1. Hi96MNPCelB2 strain streaked on LB agar plate.

Figure 2. 1% Agarose Gel

Figure 3. CelB2 Purification Diagram

Figure 4. 10% SDS-PAGE (Novex, NP0316). CelB2 Expression and Affinity Purification.

Table 1. Absorbance at 280 nm for Affinity Chromatography Elutions

Figure 5. IEX Chromatography Elution Profile

Table 2. IEX Chromatography Elutions Activity Profile

Figure 6. 12% SDS-PAGE (Novex, NP0316). Factor Xa MBP-CelB2 Fusion Digest, Buffer Exchange, and IEX Fractions with CelB2 Activity

Table 3. CelB2 Purification Table

Discussion

Observing the bands for pre-induction and post-induction from Figure 4 (lanes 2 and 3 starting from the left, respectively), it is clear that MBP-CelB2 is expressed in the post-induction lane and not in the pre-induction lane. This band is MBP-CelB2 because it is in the same position laterally as the bands in the elution fractions, which are known to be MBP-CelB2. This evidence agrees with the expected outcome as CelB2 should not be expressed pre-induction. These lanes contain many other unwanted proteins and therefore further purification steps need to be carried out. Observation of lane 4 in Figure 4 containing cell lysate shows us that cell lysis was efficient. This is evident because all the bands in this lane were darker and thicker than the post-induction lane. This gave us more protein to work with but also further justified the need for subsequent purification steps because of the increased levels of unwanted proteins. Comparing this to flow through in lane 5, it is clear that MBP-CelB2 did bind to the amylose column. This is because these two lanes show very similar band sequences. Careful analysis of the affinity chromatography elutions in lanes 7 through 11 show that MBP-CelB2 did elute from the column, but there are still some other proteins that were not separated from the solution in the column. Affinity chromatography was successful and, for the most part, purified MBP-CelB2 well. Possible experimental changes such as using a different resin in the column, maybe dextrin sepharose resin, may be an option for future experiments.

Figure 6 details the levels of recovered MBP and CelB2 before the digest, after the buffer exchange, and the IEX fractions. We did not have any post Factor Xa digest sample to analyze and therefore cannot compare the digest’s efficiency. We can compare, however, lanes 2 and 3 which correlate to before the digest and after the buffer exchange, respectively. The difference between these lanes is that the before Xa digest sample contains MBP-CelB2 along with factor Xa and other impurities whereas the after buffer exchange is cleared of impurities and we see a stronger MBP, faint CelB2, and faint factor Xa band. The MBP-CelB2 fusion might have been cleaved but our gel does not show a very strong CelB2 band, and we can conclude that we did not recover equal levels of MBP and CelB2. Possible experimental changes may include using less Factor Xa in the digest step.

Table 2 details the activity profile of CelB2 after DEAE chromatography. Fraction 9 displays the highest absorbance at 405 nm and activity according to the amount of para-nitrophenol released (.201 M). This corresponds to the band we see around 25 kDa in lane 6 in Figure 6. This faint band represents CelB2. Although it is faint, this band is still darker than other bands from the other fractions of the DEAE chromatography.

According to Table 3, it is clear that the most protein was lost after the DEAE chromatography. We also did not have the affinity chromatography elutions to compare this data to. The purification fold is greatest in the DEAE chromatography step as this is the only data we have for our table. Based on analysis of Figure 4, however, it seems that the amylose affinity chromatography step would have shown the most protein lost and highest purification fold. This cannot be known without the quantitative data we are lacking.

Overall, CelB2 isolation and purification was satisfactory. High enough levels of CelB2 were not recovered but CelB2 was recovered nonetheless. The chromatography steps seem to show the most loss of CelB2 and therefore certain changes should be made here. Using a different resin for the amylose affinity chromatography should be done in a subsequent experiment.

References

Tran, C.T.H., Nosworthy, N.J., Kondyurin, A., McKenzie, D.R., and Bilek, M.M.M. (2013). CelB and β-glucosidase

Immobilization for Carboxymethyl Cellulose Hydrolysis. RSC Adv. 3, 23604-23611.

Makwana, E. K. (2012). Stabilizing Cellulase: A Study of the Thermal Stability of CelB2, the β- 1,4 Endoglucanase from Streptomyces Lividans by Replacing Selected Charged Residues with Uncharged Structural Analogs. Thesis. Rutgers University, Web.

Sulzenbacher, G., Mackenzie, L.F., Wilson, K.S., Withers, S.G., Dupont, C., and Davies, G.J. (1999). The Crystal Structure of a 2-Fluorocellotriosyl Complex of the Streptomyces lividans Endoglucanase CelB2 at 1.2 Å Resolution. Biochemistry. 38, 4826-5833.

Sulzenbacher, G., Shareck, F., Morosoli, R., Dupont, C., and Davies, G.J. (1997). The Streptomyces lividans Family 12 Endoglucanase: Construction of the Catalytic Core, Expression, and X-ray Structure at 1.75 Å Resolution. Biochemistry. 36, 16032-16039.

Zechel, D.L., He, S., Dupont, C. and Withers, S.G. (1998). Identification of Glu-120 as the Catalytic Nucleophile in Streptomyces lividans Endoglucanase CelB. Biochem.J. 336, 139-145.

Supporting Information

1.) Figure X. Log MW vs Distance

2.) Bradford Assay

3.) Sample Calculation for Purification Table

Regression analysis of BSA standards give y= 0.0012x + 0.4314

Average concentration of combined DEAE fractions calculated from Bradford assay is 56.89 mg/mL and total volume is 2 mL. Therefore, (56.89 mg/mL)(2 mL) = 113.78 mg protein

Lysate concentration calculated from Bradford assay is 4458.67 mg/mL and total volume is 7 mL. Therefore, (4458.67 mg/mL)(7 mL) = 31,210.7 mg protein

Activity found in the activity assay for DEAE elutions is 0.0183 ΔA/min which equals (0.0183 ΔA/min)/ (18.1*0.1 cm) = 0.01 units

Activity found in the activity assay for lysate is 0.250 ΔA/min which equals (0.250 ΔA/min)/(18.1*0.1cm) = 0.138 units

Specific activity for DEAE elutions is 0.01 units/113.78 mg = 8.79 x 10-5 units/mg

Specific activity for Lysate is 0.138 units/31,210.7 mg = 4.42 x 10 -6 units/mg

Purification fold for the combined DEAE elutions is (8.79 x 10-5)/(4.42 x 10-6) = 19.89. The yield is (0.01/0.138)*100= 7.25%