Transcript Slide 1

Development of a Method for Isolation of
Phosphorylated Proteins using Alkaline Phosphatase
Dema Alniemi, Duane Mooney, Royce Wilkinson, Edward Dratz
Department of Chemistry and Biochemistry, Montana State University
April, 2011
2011 Hughes Scholars Undergraduate Biology Program
INTRODUCTION
Proteomics, the study of the proteins present in cells and their posttranslational modifications (PTMs), is an increasingly important field of
study. Proteins are responsible for most of the signaling, metabolism,
and mechanical action in cells, and PTMs control these activities. More
than 400 PTMs have been identified, the most common being covalent
attachment of small molecules to intact proteins. One such molecule
essential to many protein functions is phosphate. Addition or removal
of a phosphate group at specific sites on proteins causes changes
in chemical activity or sub-cellular location, thereby controlling
cellular activities such as gene expression, metabolism, cell death,
and initiation or termination of a protein’s activity.
Mutant, inactivated ALP has been prepared that retains its ability to bind
phosphorylated proteins, but does not take off the phosphate. We plan
to use this mutant ALP as an affinity matrix for isolating
phosphoproteins.
The expression plasmid pEK154 containing either the WT or the
mutated gene was transformed into E. coli strain SM547.
Transformants were isolated and the DNA sequence of the region
of interest was determined by the Nevada Genomics Center,
using a sequencing oligo as primer which hybridized starting at
nucleotide #95.
Mutation of wild type ALP to S102L at active site was created
through use of oligonucleotide primers with the desired mutation
on the plasmid in a Mutant Strand Synthesis Reaction.
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Wild Type
Mutant
Altered phosphorylation has also been found to play a role in many
diseases such as diabetes and cancer. With much of our world’s
population being affected with such diseases, work to more efficiently
and effectively isolate phosphorylated proteins and to determine
changes in phosphorylated proteins in health and disease is becoming
increasingly crucial for scientific and clinical applications.
My research has been focused on developing a new method for
enrichment and isolation of phosphorylated proteins by the use of the
Escherichia coli (E. coli ) alkaline phosphatase (ALP) enzyme. ALP is a
highly non-specific phosphatase that hydrolyzes the bond between a
phosphate group and its attached molecule.
DISCUSSION
METHODS
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Thr
Asp
Ser
Ala
Ala
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Thr
Asp
Leu
Ala
Ala
WT ALP was purified from the periplasm of stationary-phase
cells. The crude periplasmic proteins were heat denatured at 80C
for 10 min. After centrifugation, the soluble ALP was precipitated
in 85% ammonium sulfate. After dialysis, ALP was further purified
by ion exchange on Q-sepharose.
Enzyme purification was monitored at each step by enzymatic
assays, and by SDS polyacrylamide gel analysis. Specific
activities were calculated based on moles of PNPP substrate
hydrolyzed/min/mg of protein.
Figure 3. Only a single amino acid change at the active site
was made; mutation of nucleotide C371 to T371 leads to the
substitution of leucine for serine at amino acid position 102.
RESULTS
The desired mutation displayed in Figure 3, S102L, occurred as
planned; therefore we were able to work with the mutant form of
ALP and perfect the procedures used. However, the mutant
enzyme seemed to be lost among the steps of purification, as it
was lacking in the proper area as compared to the WT enzyme.
Early work led to the idea that the mutation was successful, as
DNA was present during the mutagenesis experiment (data not
shown), and initial DNA sequencing efforts gave positive mutation
results. Activity may have been lost during purification or in a yet
undetermined preliminary step.
The silver-stained SDS gel (Fig. 5A) demonstrated that osmotic
shock and heat treatment markedly increased the purity of ALP.
The vast majority of proteins become insoluble when exposed to
high temperature, but ALP remains soluble while maintaining its
enzymatic activity, making this simple method a major step in
purification. Figure 5A also shows that the mutant enzyme was lost
or was not well expressed after purification. Further testing needs
to be conducted to determine the reasons behind such a result.
CONCLUSIONS
Mutation of the wild type ALP through use of a Mutant Strand
Synthesis Reaction was successful, as observed from DNA
sequencing results. Methods and protocols used in purification of
wild type ALP as well as assays to determine purity and activity
were therefore used for both the mutant and wild type ALP
simultaneously. Results shown are for mutant and wild type
alkaline phosphatase.
Transformant #
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Figure 4. Agarose gel showing
plasmid DNA with the target gene
after mutation. Plasmid supercoils (SC)
and nicked circles (NC) are indicated.
SC
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Std
NC
The development of a more powerful and effective method for
enriching phosphoproteins promises to be unique in this field of
research. Effective methods now work at the peptide level and
require protease digestion and “uncoupling” of different PTMs in
different parts of proteins. The enriched phosphorylated proteins
can be separated intact on 2D gels to more clearly show changes
in different PTM isoforms.
The ability to better track changes in protein phosphorylation
promises to be extremely beneficial for advancing scientific and
clinical research. If this method proves to be successful in
enriching phosphorylated proteins from their unphosphorylated
counterparts, large advances can be made in the field of
proteomics, as well as broadly in biomedical research. We have
succeeded in developing methods that are foundational for the
ultimate goal of this project, and anticipate reaching our target
purpose in the near future.
A
Figure 1. Structure of alkaline phosphatase. The wild type
E. coli alkaline phosphatase efficiently removes the phosphate
from phosphoserine, phosphothreonine and phosphotyrosine
sites on proteins.
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FUTURE WORK
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B
Figure 5-A: Silver stained SDS-PAGE gel of WT and
mutant ALP. Lanes from left to right show select steps during
increasing salt wash concentration purifications for both WT
and mutant forms of the enzyme. Lane #1. WT .05M; #2. WT
.075M (1); #3. WT .075M (2); #4 WT .10M (1); #5 WT .10M
(2); #6 WT .10M (3); #7 WT .10M (4); #8 MUT .05M; #9 MUT
.075M (1); #10 MUT .075M (2); #11 MUT .10M (1); #12 MUT
.10M (2); #13.10M (3); #14 MUT .10 (4); #15 Standard
Li et al (2009) reported that the S102L ALP mutant binds phosphorylated
proteins with high affinity, but fails to remove the phosphate residue.
Currently available methods for purifying phosphorylated proteins suffer
from various shortcomings. We plan to use ALP(S102L) as a unique
means to purify phosphorylated peptides and proteins. Coupled with
powerful differential detection methods for revealing changes in
phosphorylation developed in our lab, this method is expected to provide
insight into the workings of cells as well as provide information as to why
certain diseases develop and affect humans.
For my research, the mutation S102L was created in ALP by site-directed
mutagenesis. Due to the periplasmic location and heat stability of
ALP in E. coli, it is easy to purify in high yield. The wild type protein
was expressed in E. coli, purified from the periplasmic space, then
enzymatic activity and purity were determined as a first major step.
B
Amount Protein in Selected Steps During Column Purification
Kinetic Measurements of Enzyme Activity
600
Total protein (ug)
Figure 2. Close-up view of the ALP catalytic site. A: Catalytic
center of WT ALP showing interaction between S102 and the
phosphate. B: Altered structure of the catalytic center of ALP S102L
with a docked phospho-peptide.
ALP enzyme activity was calculated from the rate of
appearance at 405nm absorbance due to hydrolysis of
p-nitrophenol phosphate (PNPP) to p-nitrophenol (PNP)
and phosphate, with an extinction coefficient for PNP of
18,000.
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M
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M
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Successive salt wash steps
Figure 5-B. The amount of protein was measured in the indicated
salt washes during Q-Sepharose chromatography.
The Km of PNPP binding to ALP was found to be 3.1uM
which is essentially equivalent to the reported value of
3.6uM.
Future work requires demonstrating a means to covalently bond
the mutant ALP to glass beads, providing a simple way to use
this reagent. Ultimately, 2D gels and mass spectrometry will be
utilized to characterize changes in phosphorylated proteins in
health and disease through this method.
REFERENCES
Li, W., et al. Development of a universal phosphorylated peptidebinding protein for simultaneous assay of kinases.
Biosensors and Bioelectronics, 2009, 24:2871-2877.
Tarrant, M., and Cole, P. The Chemical Biology of Protein
Phosphorylation. Annual Reviews in Biochemistry, 2009,
78:797-825.
Wilkinson, R., and Peters, T. Biochemistry and Molecular Biology
Methods, 2010.
http://www.chemistry.montana.edu/~martint/
BCHM444/Syllabus_S10.pdf
ACKNOWLEDGMENTS
I’d like to thank Dr. Edward Dratz and his lab for working with me,
with a special thanks to Duane Mooney for guiding me through my
project with knowledge, patience, and kindness. Thanks as well to
Martha Sellers and the Hughes Undergraduate Biology organization.
The Hughes Scholars Program is funded by the Howard Hughes
Medical Institute.