purification project
TRANSCRIPT
Purification of Streptomyces lividans endoglucanase CelB2 expressed in E.Coli strain
BL21(DE3)
Mujtaba M. Qureshi
Department of Biochemistry
Rutgers School of Environmental and Biological Sciences
732-649-9070
Abstract
Cellulose, a plant polymer of D-glucose (bonded together by B-1,4-glucosyl linkages), is
the main component of plant cell walls and the most abundant protein in the world. It has largely
been considered as a possible alternative as a fuel source. We induce transformation of
mutants of Streptomyces lividans endoglucanase CelB2, an enzyme that hydrolyzes cellulose,
into the E.Coli strain BL21(DE3) in order to recover the endoglucanase enzyme, which can be
used to make cellulose a more viable alternative as a fuel source. The CelB2 mutant has amino
acid residues that are substituted in order to make a more thermally stable enzyme by making
the protein into an uncharged/neutral molecule. The E.Coli strain BL21(DE3) is better suitable
for transformation and protein expression and will upscale the amount of protein recovered. We
hypothesize that CelB2 will be recovered and the specific activity will increase several fold as
the protein is purified. We express CelB2 as His6MBPCelB2 fusion in order to separate the
enzyme from all other proteins from the cell by the use of amylose affinity chromatography. We
use Factor Xa, a protease, to cleave MBP from CelB2, and then use an Anion-Exchange
column (DEAE Sepharose) to further purify CelB2 from the solution. We use an activity assay
along with SDS-PAGE to measure and track the presence and concentration of CelB2. We use
the Beer-Lambert law to calculate [CelB2] to be 2.286uM. Although specific activity increased
and CelB2 was purified, only low levels of CelB2 were recovered. Future studies should aim to
further improve the procedure of CelB2 recovery, potentially through more stable variants of
CelB2 fusion protein or a better mutant of CelB2 itself.
Introduction
Cellulose, a plant polymer of D-glucose (bonded together by B-1,4-glucosyl linkages), is
the main component of plant cell walls and the most abundant protein in the world. It has largely
been considered as a possible alternative as a fuel source. As a stable molecule, the hydrolysis
of cellulose requires high temperatures and rough chemical means. Alternatively, cellulose can
be hydrolyzed by enzymes, like that of endo-1,4-B-glucanase: an enzyme that hydrolyzes the B-
1,4-glucosyl linkages at random. Through the use of such enzymes, cellulose can be converted
and fermented into other fuel sources, such as ethanol (Huang et al., 2005). The uses for a
thermally-stable endoglucanase/enzyme can thus be largely important in real world application
to make cellulose a more viable fuel source for energy.
We focus in this study on mutants of Streptomyces lividans endoglucanase CelB2, a
more stable enzyme. 4 CelB4 variants were designed: D108N, E149Q, D175N, and D178N.
The variants D108N,D175N, and D178N had the aspartate residue at their respective locations
replaced with aspargine, and E149Q had glutamate replaced with glutamine. The amino acid
substitutions were done to make CelB2, a polar charged molecule protein, into a polar
uncharged/neutral molecule. The changes produce little disruption to the native conformation
and make CelB2 a more stable protein.
In this study, we use a the E.Coli strain BL21(DE3), a chemically competent strain that is
efficient in transformation and thus protein expression. It has a transformation efficiency of 1–5 x
107 cfu/µg pUC19 DNA and is deficient in endogenous proteases Lon and OmpT, which are
responsible for degradation of proteins in vitro systems. It is also resistant to phage T1 (fhuA2)
and expresses T7 RNA polymerase. We aim to induce transformation of this model organism
with plasmid for Streptomyces lividans endoglucanase CelB2 and to further purify and recover
the protein produced.
We use IPTG (Isopropyl β-D-1-thiogalactopyranoside), a compound that triggers
transcription of the LAC operon. It is used to induce constant expression of the CelB2 gene in
order to maximize CelB2 protein. We use pMALc4CelB2 as our expression vector in order to
create a protein fusion of maltose binding protein (MBP) and CelB2. We use an amylose resin
column as a protein separation technique to purify the fusion protein from the bacteria cells by
binding of the maltose binding protein to the amylose resin. We hypothesize that this stable
strain of CelB2-MBP fusion protein will be expressed in the bacteria and that specific activity of
CelB2 after purification increases several fold after every protein separation assay conducted.
Methods
Expression
CelB2 was expressed in E.coli BL21(DE3) cells as described previously (Makwana,
2012). The following protocol was adapted from the QIAGEN test prep protocol. E.coli was
streaked from a 50% glycerol stock onto an LB agar plate with ampicillin and then incubated at
37°C for 18 hours. Result is shown as Figure 1 in supporting information. Next, a single colony
was isolated and inoculated into a 5mL LB starter culture that contained 10uL ampicillin. The
starter culture was incubated at 37°C for 18 hours, on a shaker set to 250 rpm. The culture was
then added into a 500mL LB expression medium. The expression medium contained 500uL
ampicillin and 0.2% glucose and was grown until an OD600nm of 0.6 was reached. A 200uL
aliquot of this pre-induction sample was saved for future SDS-PAGE analysis. Induction was
then carried out on the sample via addition of 500uL of IPTG and then grown for 3 hours. A
200uL aliquot of this post-induction sample was saved for future analysis.
Plasmid Purification and Restriction Analysis
Using Qiagen Miniprep kit, the manufacturer’s protocol was followed to purify plasmid
pMALCelB2 from BL21(DE3) starter culture. DNA was eluted from the QIAprep column with
40uL of dnase-free H2O. Cells were then harvested by centrifugation at 4,000rpm in 4 steps of
5 minutes each for a total of 20 minutes at 4°C. The pellet formed from 500 mL of the
expression medium was stored at -20°C.
30uL of the expression plasmid DNA that was taken was mixed with 5uL of buffer and
20uL of DI water. 5uL of EcoR I restriction enzyme was added to this sample, and then the
sample was incubated at 37degrees Celsius, digested for 1 hour at 37°C. An aliquot of this
sample was taken and used as the ‘single cut’ for gel electrophoresis. 5uL of HindIII restriction
enzyme was added to the original sample, and then incubated for 1 hour. This sample was used
as the ‘double cut’ for gel electrophoresis. 1% Agarose Gel Electrophoresis was prepared and
run on 100V for 30minutes, and then visualized under UV Light.
Cell Lysis
250mL of expression pellet was re-suspended in 5mL of 20mM Tris buffer. Then,
3.75mg of Lysozyme was added to 15mL of the solution and kept on ice for 20 minutes. Next,
the cells underwent cell lysis through sonication, on ice, for a total of 8 minutes in 10second
pulses. 300uL of protamine sulfate was then added and incubated for 5 - 15 minutes. The
solution was then centrifuged at 10,000 rpm for 30 minutes. A 20uL aliquot of the supernatant
was taken and placed into a 1.0mL centrifuge tube for future SDS-PAGE. A 100uL aliquot of the
supernatant was also drawn and saved for future activity measurement. The remaining
supernatant, 17.9mL, was transferred into a 50mL conical tube and stored at - 20°C.
Amylose affinity chromatography
An Affinity Chromatography was setup to purify the protein, since the maltose binding
protein fused with CelB2 is able to bind with the amylose resin in the column. The preparation
entailed by mixing 3 mL of amylose resin and 10mL of Tris buffer. The solution was then
centrifuged for 3 minutes at 500rpm. Tris buffer was then decanted. The cell lysate supernatant
was mixed with 3mL of amylose resin and then incubated for 1 hour, while shaking, at 4°C. Half
of the column was filled with tris buffer. Then, the resin-lysate mixture was added to form a bed
in the column. 30 minutes later, most of the buffer was drained, leaving some buffer to keep the
resin-bed wet. Flow through was collected. Next, 25mL of the tris buffer solution was poured in
as wash, and 25mL of wash was collected as well. Then, 7mL of the elution buffer, tris buffer-
maltose solution, was poured in, and elution fractions were collected in 1mL quantities.
The elution fractions were measured using a Nanodrop. The absorbances at 280nm
from elutions 1 through 6 were as follows: 0.541, 0.941, 0.267, 0.060, 0.023, 0.032. Most of the
protein were thus in elutions 1 to 3. An SDS page was carried out as well for all 6 elutions, flow
through, and wash.
Factor Xa digest and Buffer Exchange
Affinity chromatography elutions 1 to 3 were combined. 1ug of Factor Xa was added for
every 50ug of protein in the combined elutions solution, which was estimated assuming average
protein concentration of 0.5mg/mL. The solution was gently mixed (pipetted up and down), and
then incubated overnight at 23°C. 20uL aliquots were taken before and after factor Xa addition
for SDS-PAGE.
The next day, a buffer exchange procedure was carried out using a Microsep 3K
centrifugal device. The filter was pre-rinsed with di H2O, centrifuged at 7,500g, and then the
protein solution was pipetted into the filter device and spun at 7,500g. Piperazine buffer was
was reconstituted and re-concentrated and then spun again (30 minutes). This step was
repeated four times. An aliquot of 20uL was taken for SDS-PAGE as a post-buffer exchange
sample.
Ion Exchange Chromatography and Buffer Exchange
Anion Exchange Chromatography was carried out using DEAE-sepharose as resin.
Buffer used for wash was 20mM piperazine buffer, pH 6.0. The column was washed with 5
column volumes for a total of 30mL. A salt gradient of 0mM to 700mM NaCl was set up, and
fractions were collected at 1mL intervals, for a total of 38 elution fractions. Absorbance at
280nm was measured for each fraction.
A buffer exchange procedure similar to the previous mentioned buffer exchange was
carried out, constituting sodium phosphate buffer in place of piperazine.
End point activity assay and Bradford Assay
The activity assay was performed for the elution fractions, pre-digest, and the post-IEX
samples. 100uL of 1M substrate (pNPC: para-nitrophenyl -D-cellobioside ) was mixed with
100uL of protein containing fraction and incubated at 50°C for 30 minutes. Then, 0.1M NaOH
was added to halt the reaction. The solution was diluted with 800uL H2O and then absorbance
was read at 405nm. Calculations were done using extinction coefficient at 405nm = 18.1
mL/µmol/cm, in order to obtain concentration of [pNP] released.
A standard Bradford assay was carried out using Thermofisher’s Coomassie Protein
Assay Kit, with BSA protein as the standard, in order to measure concentration of protein in pre-
digest sample and post-IEX chromatography sample. Incubation for reaction occurred at room
temperature for 10 minutes. Absorbance was measured at 595nm.
Protein Concentration & Extinction Coefficient Determination
Absorbance at 280nm was measured for the protein sample after ion exchange
chromatography. Using Beer-Lamberts law, CelB2 concentration was obtained.
Using ExPASY Bioinformatics Resource Portal program ‘ProtParam’, the following
information was obtained using the Amino acid sequence for CelB2, which was later used in the
Beer-Lambert calculation for CelB2 concentration:
Number of Amino Acids 225
Molecular Weight 23832.2
Theoretical pI 4.33
Extinction Coefficient 54680 M-1 cm-1
Table 1: CelB2 information from ProtParam. Extinction coefficient is found.
Results
A restriction analysis was performed in order to see whether or not the E.Coli had
undergone transformation, making sure that the DNA insert was successfully integrated into
plasmid. This was tested through restriction digest, and the results were viewed through gel
electrophoresis. The CelB2 insert would be found between the last two bands on the DNA
marker (between 500BP and 1000BP). We expected to see no bands in this area of interest on
lane 2 (uncut) but a heavy band on top. For lane 3 (single cut), we expected a single band of
lighter weight on top but still no band in this area of interest. For lane 4 (double cut), we
expected one band similar to lane 3 except less thick, and one band in the area of interest at
lane 4 (double cut). The appearance of the second band on lane 3, which was not expected,
leads us to believe that there was an error that occurred during the experiment; the results are
thus inaccurate. Since the expected results were not found, the plasmid purification did not work
as expected. Lane 4 did have a thick band at the top and there is a light band at the area of
interest, so we are able to say that there is a chance MBP-CelB2 is expressed and to proceed
with the experiment. If MBP-CelB2 and CelB2 are found in later steps, a possible explanation
behind the error in lane 3 could be that there is simply a contamination in the well. There is also
a ‘wave’ present on the gel which signifies errors in cooling.
Figure 1: Plasmid restrictions of pMALc4CelB2 with EcoR I and Hind III were run on a 1%
agarose gel electrophoresis at 100V for 30 minutes. The first lane is a DNA ladder/marker. The
second lane is the uncut plasmid. The third lane is the single cut plasmid via EcoR I. The fourth
lane is the double cut plasmid (EcoR I and Hind III). The plasmid lanes were loaded on with
Orange G loading dye.
Based on the SDS-PAGE in Figure 2, the MW for CelB2-MBP was estimated to be
71.8kD using the equation and Rf values of the unknown protein as shown in Figure 3. This is
slightly different than what the estimated value would have been (65kd = 23kD [celB2] + 42kD
[mbp]). The R2 value isn’t at 0.99+ and thus may show that measurement errors for Rf values
could attest to the difference in 7kD reported. Changing the percent of the gel might help,
increasing it, to better create a linear line of fit.
Looking at the gel in Figure 2, pre-Induction had fewer proteins, and post-induction had
more proteins. Specifically, post-induction had CelB2 expressed. There is a thick band at the
same estimated MW value of 71.8kD. Comparing cell lysate and flow through, we can’t tell if
CelB2 is in those lanes in the gel or not since there are a large number of proteins all
throughout. Otherwise, it seems that CelB2 had successfully eluted from the column, as seen in
our elution fraction lanes.
The values reported in table 2 for absorbances of 280nm show that the absorbances of
the elution fractions correlate with the bands shown on the SDS-PAGE in Figure 2. The highest
280nm absorbancies were for elutions 1 - 3, which are also the thickest band for MBP-CelB2
fusion as well.
Figure 2: Criterion 4 to 12% SDS-PAGE (Novex, NP0316) analysis of CelB2 expression &
CelB2 affinity purification. Lane 1 is a Molecular Weight marker Precision Plus Protein (BioRad,
161-0375). Lane 2 is pre-induction. Lane 3 is post-induction. Lane 4 is elution 1 from affinity
chromatography (Amylose resin). Lane 5 is elution 2. Lane 6 is elution 3. Lane 7 is elution 4.
Lane 8 is elution 5. Lane 9 is elution 6. Lane 10 is BL21(DE3) E.coli cell lysate. Lane 11 is the
column’s wash. Lane 12 is the column’s flow.
Figure 3: Plotted log mw vs Rf for the protein markers to estimate mw of MBP-CelB2. R^2 value
is 0.978. Equation obtained is y=-1.441x + 2.397
A280nm for Affinity Chromatography Elutions
Elution 1 Elution 2 Elution 3 Elution 4 Elution 5 Elution 6
A280nm 0.541 0.941 0.267 0.060 0.023 0.032
Table 2: A280nm for affinity chromatography elution fractions
The results from the Ion Exchange Chromatography are shown in Figure 4. The fractions
with the highest A280nm values were further analyzed on SDS-PAGE and CelB2 activity assay,
since high A280nm only correlates to a large amount of overall protein, not CelB2 specifically.
The elution fractions that were selected to be analyzed were: 14, 17, 21, and 26.
Figure 4: IEX Chromatography Elution Profile
Figure 5 (SDS-PAGE) tested the aliquots before factor Xa cut, after factor Xa cut, after
buffer exchange, the flow throughs from the IEX column, as well as the elution fractions. Lane
11 tested elution 38 as well, since it had a high A280nm value. The results should show CelB2
around 25kD in lane 3 (post-digest) as well as the lane for the fraction it eluted out on (between
7 and 11). For lane 2 (pre-digest) it should show an MBP+CelB2 fusion protein band (MW
62kD) as well a little above the 50kD band, which is visible. Although the bands for MBP (42kD)
are visible in lane 3-5 as expected (post-factor Xa, buffer exchange, and flow through), the
CelB2 bands at 25kD are not visible at all.
Figure 5: 12% SDS Page. Lane 1 = Ladder, Lane 2 = before factor Xa cut, Lane 3 = After factor
Xa cut, Lane 4 = After buffer exchange. Lane 5 = flow through 1, Lane 6 = flow through 2, Lane
7 = elution 14, Lane 8 = elution 17, Lane 9 = Elution 21, Lane 10 = 26, Lane 11 = Elution 38.
Despite the results for the elution fractions shown in figure 5, the CelB2 activity assay
(table 3) was successful and shows activity. Table 3 shows that elution #26, labeled fraction #4,
has the highest concentration of pNP, and thus the highest activity of CelB2 based on this
activity assay, since every molecule of p-nitrophenol present means that one molecule of p-
nitrophenyl -D-cellobioside was broken down by CelB2. When combining elution fractions,
elution #26 was used, along with the two elutions around it (#25 & #27). A280 nm previously
showed highest absorbency for elution #14, but the activity assay shows that the protein
concentration was lowest in CelB2 for elution #14.
Additionally, since the activity assay (table 3) shows that CelB2 is present even though
CelB2 bands were not seen on the gel in figure 5, this helps us better understand the results
reported by the SDS-PAGE. CelB2 is present in the sample elution fractions, but the reason the
CelB2 band doesn’t show up on the SDS-PAGE may be because the concentration is far too
low. The calculations in table 3 support this possibility, since the concentrations determined are
less than 1umol. Because there is far more MBP recovered than CelB2 and their concentrations
are not comparable, there is a chance that CelB2 crashed out of solution. This step of the
experiment needs to be fine-tuned to produce comparable concentrations. Using a different
protease may be a solution.
Fraction # A405nm [pNP],umol
1 (14) 0.006 (0.006)/(18.1*1)= 0.0003/0.2 = 0.00165umol
2 (17) 0.026 0.026/(18.1*1) = 0.001436/0.2 = 0.00718umol
3 (21) 0.030 0.030/(18.1*1) = 0.001657/0.2 = 0.00828umol
4 (26) 1.075 1.075/18.1 = 0.0539/0.2 = 0.29696umol
Table 3: IEX Fraction #, Absorbance at 405nm, concentration of p-nitrophenol (pNP) released
(extinction coefficient e405 = 18.1 mL/µmol/cm)
Results for the Bradford assay (figure 4) and the activity calculations (table 4, figure 5)
are shown below. Specific activity increased from pre-digest to IEX purification by 64 fold. An
increased specific activity was expected, since specific activity shows activity of enzyme in a
protein mixture, essentially showing the purity of the solution with that enzyme. The sample
became more pure as IEX was used, and the specific activity data reflects this.
Figure 4: Standard Curve for Bradford Assay, measuring A595nm
A595nm X = (A595-0.5) /0.0008 Converted to mg
Pre-Digest(Factor Xa) 0.677 221.25 ug/mL 221.25ug/mL *
1mg/1000ug =
0.22125mg/mL
Ion-Exchange
Absorbance
0.529 36.25ug/mL 36.25ug/mL *
1mg/1000ug =
0.03625mg/mL
Table 4: Calculations for [CelB2] in mg/mL for pre-digest and post-IEX samples, determined
using the Bradford Assay and the Bradford standard curve.
Procedure Total protein,
mg = total
volume (mL)
x protein
concentration
(mg/mL)
Total activity,
units
(experimental)
At 405nm
Specific
activity,
units/mg
protein
(calculate)
Yield, % Purification
fold
Amylose affinity
chromatography
(4 fractions) =
Pre-digest
4mL * .22125
mg/mL =
0.885mg
0.181 –
0.007(control)
= 0.174 units
0.174/0.885
= 0.1966
units/mg
100 1
DEAE Ion
exchange
chromatography
(3 fractions)
.300mL *
.03625
mg/mL =
0.01875mg
0.243 -
0.007(control)
= 0.236 units
0.236 /
0.01875 =
12.586
units/mg
0.236/0.174
*100 =
135.63%
12.586/0.1966
= 64.0183
Table 5: CelB2 purification: total protein, total activity, specific activity. Purification fold from pre-
digest of CelB2 solution to DEAE ion exchange chromatography is shown. Total protein and
specific activity are calculated.
We measure the A280nm value of the post-IEX sample of CelB2, and we use ExPASY
Bioinformatics Resource Portal program ‘ProtParam’ to determine the extinction coefficient for
the modified CelB2 in order to calculate the concentration of CelB2. We obtain 2.286uM of
CelB2.
A280 Path Length Extinction Coefficient [CelB2]
0.125 1cm 54680M-1 cm-1 (0.125)/(1*54680)=
2.286x10^-6 M = 2.286uM
Table 6: CelB2 concentration is determined using the Beer-lambert law, by use of Absorbance
at 280nm of the final protein solution purified from IEX.
We were unable to determine any information on the lysate sample since the sample
was lost, so we are unable to determine at what step the most protein was lost. Based on the
activity assay though, the purification fold was large (64x) from pre-digest to post-IEX
chromatography. Although this may support the idea that CelB2 was not lost greatly between
the steps, the total protein was far less for the IEX sample than it was for the pre-digest sample,
so a clear conclusion cannot be made. It is clear that not all of CelB2 was lost between the
steps since the activity fold showed an increase and not a decrease.
Overall, the protocol for expression was successful, but there were issues in CelB2
purification/recovery. There are too many steps where CelB2 can be lost, either significantly or
completely. Instead of needing to purify CelB2 through several separation techniques, it may be
more efficient to replace MBP as the protein that is fused with CelB2. It was after the fusion
protein was digested with Factor Xa that we saw a significant loss of CelB2, and that may have
been because CelB2 crashed out of solution. Testing different fusion proteins would be a good
way to find out how to best solve the issues in this step of the experiment. Trying different
proteases or incubating the aliquots and solutions at a different temperature (versus 23 degrees
Celsius) may solve this issue as well. Previous research had been conducted to make CelB2
more thermally stable as well (Makwana, 2012). Creating a new mutant of CelB2 where it is
more thermally stable by itself may improve recovery as well and would be a good future study.
References
Ekta K Makwana (2012).
Stabilizing Cellulase : A study of the Thermal Stability of CelB2, the β1,4-Endoglucanase from
Streptomyces lividans by Replacing Selected Charged Residues with Uncharged Structural
Analogs
Huang, Y., Krauss, G., Cottaz, S., Driguez, H., Lipps, G. (2005)
A highly acid-stable and thermostable endo-β-glucanase from the thermoacidophilic archaeon
Sulfolobus solfataricus
Biochemical J. 385, 581-588;
Supporting Information
Figure 1: E.coli cell culture in LB agar. Colonies had formed well.
Figure 2: CelB2 Purification scheme