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Mutagenesis and Overexpression of DNase for Single Molecule Studies
Denise Der
University of California, Irvine
IM-SURE Program, Summer 2007
Mentor: Philip Collins, Department of Physics and Astronomy, UCI
Collaborator: Gregory Weiss, Department of Chemistry, UCI
Graduate Students: John Coroneus, Jorge Lamboy, Issa Moody
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Abstract
Previous studies have nonspecifically attached a single protein to a carboxylate-functionalized
carbon nanotube. In this study, a single enzyme will be covalently and site-specifically attached
to a nanotube in a nanocircuit, and will be electronically monitored in real-time. The
investigation will be accomplished by (1) expressing and purifying four different DNase E9
mutants, (2) attaching them independently to the nanotube through cysteine chemistry, and (3)
watching for changes in conductance between the variant DNase mutants as it hydrolyzes DNA.
Ultimately, the biosensor device will be used to elucidate kinetic information of a single protein.
Key Terms

Colicin: antibacterial protein carried on a plasmid, which is composed of three globular
domains: one responsible for binding to the receptor of the cell, a second responsible for
translocation into the cell, and a third responsible for killing the cell by cleaving DNA or
RNA

Column Chromatography: a method used to separate protein based on their different
properties
o
Ion exchange chromatography: a purification technique based on the pI of the
protein
o
Affinity chromatography: a purification technique based on biological interactions
such the amino acids of a protein coordinating to the metal in a column
o

Size exclusion chromatography: a purification technique based on the mass
DNase: the 15kDa enzyme that catalyzes the hydrolysis of DNA; specifically in this
study, the DNase refers to the endonuclease domain of the colicin E9

Guanidine hydrochloride: a denaturing agent used to elute the DNase

Immunity protein: the protein that protects the cell from the DNase’s cytotoxic activity
by binding to the dnase domain of colicin E9 (the immunity protein is specific to and has
a high affinity for the DNase; the KD is ~ 10-14)1
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Introduction
Integrating nanotechnology with biology could allow the detailed study of biological
systems at the single molecule level. For example, rather than studying the dynamics of proteins
in the traditional, ensemble manner, nanotechnology could uncover how an individual protein
interacts with its surrounding environment including substrates and regulators. Isolation of a
single protein can be achieved by covalent attachment to a carbon nanotube, functionalized with
only one site for attachment. Placing this system in a nanocircuit provides a way to electrically
monitor the protein as it dynamically changes its conformation. The resultant biosensor device
could help researchers gain a better understanding of proteins.
Previously, proteins have been immobilized onto a functionalized carbon nanotube;
however, the attachment, whether through hydrophobic interactions or covalent attachment, was
nonspecific. For instance, Au-coated streptavidin was attached to a functionalized single wall
carbon nanotube after it was chemically treated with N-ethyl-N’-(3-dimethyl aminopropyl)
carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate a single carboxyl group on
the sidewall of a nanotube2. Streptavidin formed an amide linkage to the functionalized carbon
nanotube, because EDC/NHS is sensitive to the sidechain of a lysine residue. Tetrameric
streptavidin has four lysine residues per monomer. Since streptavidin has multiple lysine
residues, the exact residue attached to the nanotube and whether the positioning of this lysine
affected the performance capabilities of the nano biosensor could not be determined.
Consequently, the goal of this project is to provide site-specific protein attachment to the
carbon nanotube, which can be accomplished by cysteine-maleimide chemistry3. The
carboxylated-functionalized carbon nanotube would be exposed to EDC and, subsequently, to
NHS. Afterwards, a linker with an amine end and a maleimide end would react with the
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chemically modified nanotube to form an amide bond. Becoming maleimide-activated would
make the chemically-treated carbon nanotube reactive toward free thiols (R-S-H), which happens
to be the sidechain of a cysteine residue. The target protein would be genetically engineered to
present only one cysteine residue either at one of the terminal ends or within the protein itself.
The free thiol of this cysteine residue would then react with the maleimide through a 1,4-Michael
addition.
Because covalently attaching a protein to a carbon nanotube in a nanocircuit provides a
new way to electronically visualize a protein in real-time, a well-studied protein without any
cysteine residues must be used. The DNase domain of colicin E9 is the ideal protein for this
study since the wild-type sequence not only has no cysteine residues but also the kinetics of this
enzyme have been thoroughly studied4,5,6. A metalloprotein, DNase, can only function with a
divalent cation cofactor bound. The metal bound to the DNase affects its activity because a
transition metal such as nickel or cobalt yielded higher activity than other metals such as
magnesium5. The reason is transition metals created more nicks in both strands of dsDNA while
the other metals just introduced breaks on one strand. To examine DNA cleavage, previous
studies held the concentration of the DNase constant while varying the concentration of the
divalent cation. A large number of molecules were used to produce the overall effect of sheared
DNA. The mechanistic basis and dynamics of the process could not be determined using this
traditional method. Therefore, the goal of this project is not only to be able to elucidate kinetic
information such as the turnover number, but also to identify key mechanistic events and their
time dependence. Since DNase in this biosensor device is sensitive to its surrounding
environment, by carefully watching the conductance, an individual DNase can be monitored and
single events pinpointed as the surrounding environment changes (for example, after the addition
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of substrate). The biosensor device contrasts the old method of viewing enzymes, which only
examines average effects, typically looking at the starting and ending points.
Significantly, the biosensor device can not only be used to study the behavior of
unknown proteins, but could also potentially be used as a pharmaceutical device as well.
Currently, many drugs designed are structure-based. The crystal structure of a protein must be
known so that computational studies can be performed to scan through libraries of drugs to see if
the drug would bind to and inhibit the activity of the protein’s active site. Proteins, however, are
not rigid molecules; they are dynamic. If an enzyme is constantly changing its conformation,
then the drug might not have a chance to interact with the active site. This device could be an
effective way to test the efficacy of a drug, which might be seen with a drop in conductance
signifying the activity of the enzyme has been stopped. This could be tested in this study by
introducing the immunity protein or ethylenediamine tetraacetic acid (EDTA), which chelates
bivalent metals, to the system. The immunity protein would bind to the DNase and prevent it
from hydrolyzing DNA in this fashion while EDTA would chelate to the divalent cation and stop
the DNase from cleaving DNA. Regardless, the conductance change should be similar in both
cases, which would illustrate the potentiality of using this device for drug discovery and testing.
Materials and Methods
Site-directed Mutagenesis using Quikchange Protocol
The plasmid pRJ353 containing the DNase domain of colicin E9 gene and its immunity
protein gene in the pET21d vector was provided by Dr. Chris Penfold of the University of
Nottingham. Mutagenic primers (see Table 1) were designed by me and ordered from MWG
Biotech. For each mutation, two separate reactions initially took place: one reaction carried the
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forward primer while the other carried the reverse primer. Each reaction mixture consisted of the
2.5 μl 10x Pfu buffer, 1 μl primer, 0.5 μl pRJ353, 0.5 μl dNTP, 19.5 μl ddH2O, and 1 μl 95:5
Pfu/Taq polymerase. The reaction was polymerase chain reaction (PCR) cycled twice using this
method: 95 °C for 45 seconds to denature the DNA template, 62 °C for 1 minute to anneal the
primers to the DNA template, and 68 °C for 12 minutes to extend the primers and synthesize the
rest of plasmid. Afterwards, the two reaction tubes were mixed, so that the final aliquot volume
was 50 μl, and it contained both the forward and reverse primers. These reactions continued the
PCR cycle 16 additional times. Subsequently, the mixture was digested with the restriction
enzyme DpnI to eliminate the parental non-mutated plasmid. The remaining mutated plasmid
was transformed into heat shock competent E. coli DH5-α cells.
Colony PCR
Reactions were carried in 25 μl volumes and underwent 30 cycles of amplification with
T7 primers at 94 °C for 1 minute, 58 °C for 1.5 minutes, and 72 °C for 3 minutes. A 2% agarose
gel was run to ensure the PCR reaction worked before sending the PCR product to Genewiz for
sequencing, which would confirm the desired mutation was present.
Total cell protein
After the mutation was verified, the mutated plasmid was transformed into E. coli
BL21(DE3). A small scale culture of 20 ml Luria broth (LB), 20 μl carbenicillin, and 200 μl of
overnight culture was grown and induced with isopropyl-β-D-thiogalactopyranoside (IPTG)
when the optical density of cells at 600 nm was 0.8. After four hours, the culture was
centrifuged. The cell pellet was resuspended in phosphate buffered solution (PBS) and sonicated
before centrifugation. The samples were then examined by protein gel to verify the
DNase/immunity complex successfully expressed before growing a larger culture.
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SDS-Page Gels
Sodium dodecyl sulfate polyacrylamide gel electrophoresis provides a means to visualize
protein expression by separating them according to molecular weight. To create a 15% lower gel,
a mixture of 1.25 ml nanopure water, 1.25 ml 4x Tris base/SDS pH 8.8, 2.5 ml 30% acrylamide,
5 μl TEMED, and 33.3 μl 10% APS was made, while the upper gel consisted of 1.5 ml nanopure
water, 625 μl 4x Tris base/SDS pH 6.8, 375 μl 30% acrylamide, 2.5 μl TEMED, and 16.6 μl 10%
APS. After the gel polymerized, the samples were loaded, and ran at 150 V for 1-2 hours. The
gel was then removed, and stained with Coomassie Brillant Blue dye. The quick staining
visualization method required heating the gel while incubating in the dye for two minutes in the
microwave. The treated gel was then destained by rinsing the gel with diH2O and heating the gel
while incubating in a solution of 90% diH2O and 10% ethanol for roughly five minutes.
Acetone Precipitation
For the samples that contained 6M guanidine hydrochloride, if the denaturant was not
removed before running an SDS-PAGE gel, then the gel would not run properly. To remove the
guanindine, 1 ml of chilled acetone was added to 200 μl of the sample. Typically, when the
solution was mixed homogenously by vortex, the solution became cloudy. The solution was
incubated on ice for 10 min, which was followed by centrifugation for 10 min at14 krpm. The
supernatant was then discarded while the remaining pellet, if observed, was resuspended in 200
μl PBS. The prepared sample was then ready to run on the gel.
Protein Purification
The His-tag protocol purification protocol was adopted from Garinot-Schneider7. Two 2liter baffled flasks with 500 ml LB and 500 μl carbenicillin were inoculated with 5 ml of a starter
culture of E. coli BL21(DE3) with the mutant plasmid. Shaking and incubating at 37 °C, the
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cultures were grown to an OD600 at 0.8, overexpressed by the induction with IPTG to a final
concentration of 1 mM, and allowed to grow for another four hours. The cultures were then
centrifuged at 6 krpm for 15 minutes and stored at -80 °C.
The pellet was resuspended in 40 ml of binding buffer (5 mM imidazole, 500 mM NaCl,
20 mM Tris-HCl (pH7.5)). The bacterial suspension was sonicated and centrifuged at 16 krpm
for 45 minutes. The supernatant was then sterile filtered through a 0.45 μm membrane. This
sample was then loaded onto a nickel affinity column, washed with 40 ml of the binding buffer,
and 10 ml of the wash buffer (20 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.5)). The
DNase which is bound to its immunity protein that has a His-tag was eluted with the binding
buffer containing 6 M guanidine hydrochloride. The fractions containing the DNase were then
dialyzed against water, which precipitated most of the E. coli proteins.
Further purification steps included running ionic exchange columns. To prepare for an
anionic exchange column, the protein to be purified was dialyzed overnight against a low salt
buffer (10 mM NaCl, 20 mM Tris-HCl pH 7.5). The protein sample was loaded onto a column
carrying positively-charged functional groups, which attracted negatively charged proteins.
Because the pI of the DNase is 9.5, at a pH of 7.5, the DNase should be positively charged and
should end up in the flowthrough, not binding to the column. The other proteins that bound to the
column were gradually eluted with a high salt buffer (1 M NaCl, 20 mM Tris-HCl pH 7.5).
Similarly, to prepare for a cationic exchange column, the protein to be purified was dialyzed
against a low salt buffer (20 mM NaCl, 8 mM KH2PO4, 16 mM Na2HPO4 pH 7.2). The protein
was then loaded onto a column carrying negatively-charged functional groups, which attracted
positively charged proteins, the DNase. Bound proteins were gradually eluted with a high salt
buffer (1 M NaCl, 8 mM KH2PO4, 16 mM Na2HPO4 pH 7.2).
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Mass Spectroscopy
Preparation included mixing 9 μl of the sample with 1 μl of 10% trifluoroacetic acid
(TFA). To use the MALDI mass spectrometer, a spoonful of sinapic acid needed to be mixed
with 50% acetonitrile (ACN): 50% water. After vortexing and centrifuging the mixture, the
supernatant was discarded. The process of adding 50% ACN: 50% H2O, vortexing and
centrifuging was repeated, but the supernatant was not discarded the second time; 10 μl of the
supernatant was added to the TFA/protein mixture. Half a microliter was spotted onto a plate and
run through the mass spectrometer.
Results
Thus far, all four mutants were made successfully using
the Quikchange protocol. The two internal mutants had a serine
residue replaced by a cysteine residue—one near the active site
at position 30 while the other distal to the active site at position
49. For the amino terminus mutant, amino acids lysine and
cysteine were inserted at the beginning of the DNase while for
the C-terminus mutant, a cysteine residue was inserted at the
carboxyl terminus. (The lysine residue, right after the
methionine, was inserted into the amino mutant because it
increases the efficiency of translation8.) These mutated
Figure 1. SDS PAGE gel of the
DNase/immunity protein
complex. Lane 1, molecular
weight ladder; lane 2, post-lysis
cell pellet; lane 3, post-lysis
supernatant; lane 4, pre-lysis
supernatant
plasmids were transformed into E. coli BL21 DE3, and were shown to overexpress the desired
two proteins (the DNase and its immunity protein). See Figure 1 of the internal mutant at
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position 30. The DNase is expected to be around 15 kDa while the immunity protein is smaller
falling around 11 kDa.
We’re currently working on purification of the DNase. From the SDS PAGE in Figure 2,
eluting with 6 M guanindine hydrochloride separated the DNase from the immunity protein;
however, it was not pure. Random E. coli proteins were eluted with the DNase, but after dialysis
against water, the E. coli proteins aggregated, and precipitated out (Figure 3). However, even
though the gel appears to depict one fat band of protein at the expected molecular weight of 15
kDa, the size exclusion chromatography demonstrated that it was actually two proteins with very
close molecular weights.
Figure 2. SDS PAGE gel after purification
using a nickel affinity column. Lane 1,
flowthrough; lane 2, flowthrough after acetone
precipitation; lane 3, binding buffer collection;
lane 4, wash buffer collection; lane 5, molecular
ladder; lane 6, elution fraction 1 after acetone
precipitation
Figure 3. SDS PAGE gel after dialysis
again nanopure water. Lane 1,
precipitate from the dialysis; lanes 3-4,
different aliquots of the supernatant from
the dialysis; lane 5, molecular weight
ladder
Discussion
Unfortunately, no attachment to the carbon nanotube has been attempted thus far. The
DNase protein has to be absolutely pure before introducing it to the functionalized carbon
nanotube; if another cysteine containing protein happens to be present, it could bind to the
nanotube instead of the desired DNase. The chromatograph (Figure 4) of running the sample
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through the size exclusion shows two peaks that were merging, which gives the appearance of
two proteins having very similar molecular
weights. However, the sample was run through
the MALDI mass spectroscopy which showed
two peaks—one large peak being the DNase
around 15 kDA while the other was the small
Figure 4 Chromatogram from the size exclusion
column run. The red line is the UV line used to detect
the amount of protein collected in each fraction.
peak of 11 kDa. The expected molecular
weight of the mutated DNase is 15.11 kDa
while the actual peak was 15.104 kDa. Even though the mass spectrum gave off the appearance
that only one protein is present around 15 kDa, the other protein probably could not fly through
the mass spectrometer (meaning no peak would appear). Both anionic and cationic exchange
columns were performed in attempts to separate the proteins. However, the ion chromatography
technique did not successfully purify the DNase. The smaller protein is hypothesized to be a
fragment of the DNase, which is why it could not separate after the ion exchange runs (being a
fragment of the DNase would mean it has similar properties and thus cannot be separated based
on charge). To test the hypothesis, a protease inhibitor will be added before cell lysis to prevent
the DNase from being cut up by a protease.
After the purification of the mutant DNase has been established, the other three mutants
will hopefully follow more smoothly. Then, ensemble kinetic studies can be performed, which
would be compared to and contrasted with the conductance data extracted from attaching the
enzyme to the carbon nanotube.
Acknowledgements
I would like to thank Dr. Chris Penfold for providing us with the pRJ353 plasmid, Professor
Philip Collins for being my mentor during the IM-SURE program, Professor Gregory Weiss for
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overseeing my progress and helping me along the way, and the National Science Foundation for
funding this project.
Works Cited
1. Keeble, A.H., Hemmings, A.M., James, R. et al. “Multistep Binding of Transition Metals to
the H-N-H Endonuclease Toxin Colicin E9.” Biochemistry. 41 (2002): 10234-10244.
2. Goldsmith, B.R., Coroneus, J.G., Khalap, V.R. et al. “Conductance-Controlled Point
Functionalization of Single-Walled Carbon Nanotubes.” Science. 315.5808 (2007): 77-81.
3. Dietz, H., Bertz, M., Schlierf, M., et al. “Cysteine engineering of polyproteins for singlemolecule force spectroscopy.” Nature Protocol. 1 (2006): 80-84.
4. Walker, D.C, Georgiou, T., Pommer, A.J., et al. “Mutagenic scan of the H-N-H motif of
colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and
apoptotic endonucleases.” Nucleic Acids Research. 30.14 (2002): 3225-3234.
5. Pommer, A.J., Wallis, R., Moore, G.R., et al. “Enzymological characterization of the nuclease
domain from the bacterial toxin colicin E9 from Escherichia coli.” Biochem. J. 334 (1998): 387392.
6. Van den Heuvel, R. H.H., Gato, S., Versluis, C., et al. “Real-time monitoring of enzymatic
DNA hydrolysis by electrospray ionization mass spectrometry.” Nucleic Acids Research. 33.10
(2005).
7. Garinot-Schneider, C., Pommer, A.J., Moore, G. et al. “Identification of Putative Active-site
Residues in the Dnase Domain of Colicin E9 by Random Mutagenesis.” J. Mol. Biol. 260
(1996): 731-742.
8. Stenstrom, C.M, Jin, H., Major, L.L. et al. “Codon bais at the 3’-side of the initiation codon is
correlated with translation initiation efficiency in Escherichia coli.” Gene. 263 (2001): 273-284.
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