Protein Methods

Anu Kumar
Seattle Biomedical Research Institute
anuradha.kumar@sbri.org
Protein techniques
Protein Identification
Protein Expression
Protein Purification
Protein-Protein interactions

Application in literature
Protein Identification
Sequencing (Edman degradation)
Determine approx the first 20 AA
Centrifugation (cellular location)
1D/2D Gel Electrophoresis
Mass spectrometry
Break sample into peptides
Molecular mass is determined using mass-
to-charge ratios of ions
AA sequence can be determined
Centrifugal separation
Differential centrifugation
Sediment coefficient (mass, density and shape)
Crude separation of cell fractions
Rate Zonal (mass)
Density of particles > density of solution
Separation based on rate of sedimentation
Time sensitive
Isopycnic (density)
Density of particles < highest density of solution
Separation based on reaching equilibrium position in
density gradient
Time in-sensitive
Differential
centrifugation
Gradient
centrifugation
Gel electrophoresis
Denaturing SDS-PAGE
SDS gives uniform neg. charge
Separates proteins by size/mass
Non-denaturing
Separates based on charge and
size/conformation
Often combined with Western blotting
(using antibodies specific for proteins
of interest)
SDS-PAGE
2D gel electrophoresis
1st dimension
Separation based on pI
isoelectric focusing of zwitterions

2nd dimension
Normal SDS-PAGE
2D-GE
Difference Gel Electrophoresis
(DiGE)- quantitative
comparisons
Mass spectrometry
IDs based on mass-to-charge ratio
Samples are broken down and
analyzed
Proteins -> peptides
Able to determine seq of peptides
Database search to ID protein
Mass spectrometry
Mass spec combos
LC/MS
Liquid chromotography to separate peptides
MALDI-TOF MS
Matrix-assisted laser desorption-time of flight
Samples are ionized and flight time through
an electrified tube is measured
Tandem MS
Multiple MS measurements on a single sample
Identifies peptide sequence
Protein Expression and
Purification
Why?
Obtain pure (clean) protein
"Don't waste clean thinking on dirty enzymes
- Arthur Kornberg
Powerful experimental tool
Simplifies the system in which you are
asking a question
Confirmation of a hypothesis that is
developed in a more complex system
Protein Expression
Why over-express the protein?
Make large quantities to facilitate
purification/study
Analyze biochemical properties
Perform structural analyses
Crystallization
NMR
Identify protein interactions
Make Antibodies
Expression systems
E. coli
Prokaryotic expression workhorse
Yeast
For bacterial or eukaryotic proteins
Large amounts of protein
Insect cells
Post-translational modifications
In-vitro systems
wheat germ, rabbit reticulocyte
Purification strategies

Exploiting protein chemistry

Size/Mass

Charge

Hydrophobicity

Antibody affinity

Protein Tags

Often used in combination

Size Exclusion
Chromatography
Separation based on size of protein
Ion exchange &
hydrophobicity
Non-tagged proteins
Separation based on charge or
degree of hydrophobicity
Bound proteins are eluted with
salt containing buffers
Ion exchange
HPLC
High performance (pressure) liquid
chromatography
Sample is passed over column of
varying hydrophobic nature- more
hydrophobic particles bind tighter and
elutes later.
Eluate is analyzed by a detector
UV, refractive index, fluorescence
Can be combined with mass spec
(LC/MS)
HPLC
Affinity/Ab columns
Purify tagged proteins
Interaction between two molecules
Solid phase- immobilized on column
Mobile phase- binds while passing
over column
Buffer conditions regulate binding
& dissociation
pH, ionic strength, competing
Ab affinity column
Tagging the protein
Clone gene in frame with a
unique protein sequence or tag
Advantages
Purification
Use tag to selectively remove protein
from a complex sample
Protein visualization/tracking
Fluorescent protein tags, labeled
antibodies
Protein Tags
His
small
6 HIS residues bind to nickel columns
GST
Binds to glutathione resin/beads
S-Tag, C-myc, HA, flag
Antibody affinity columns
Protein Tags
6xHistidine binds metal chelating resin-
Cu2+, Ni2+, Co 2+
What can you do with
purified proteins?
Biochemical and functional
characterization
DNA binding, enzyme activity, stability,
etc.,
Structural analyses informs function
NMR, crystallography, circular dichroism
Study protein-protein or protein-DNA
interactions
Develop antibodies
Structural Analyses
Circular dichroism (basic 2 o
structure)
Nuclear Magnetic Resonance
Protein Data Bank- pdb.org
X-ray Crystallography
Electron Microscopy
Using structure to inform drug
design/mutagenesis
Using multiple fragment binding in an enzyme
active site to determine possible directions of
growth chemistry within the active site.
Fragment linking: X-ray crystal structure of
fragments binding at different sites of
thrombin

S2S4 sites S1 site Structure of the final

(IC50=12M) (IC50=330M inhibitor (IC50=3.7nM)
)
Protein-protein
interactions

Two hybrid system

Co-immunoprecipitation
Surface plasmon resonance
Protein arrays
Protein crosslinking
FRET- fluorescence Resonance Energy
Transfer
Two hybrid systems
Used to identify two interacting
proteins
Uses bait protein to fish for
interacting proteins
Binding of fish protein leads to
selective gene expression
Reporter gene expression, ie -gal
Antibiotic gene expression
Bait and Prey proteins are
tagged with yeast
transcription factors:
Immunoprecipitation
Specific Ab binds protein in solution
Solution is eluted over Protein A column
Protein is eluted from Ab
Co-IP
Also allows for study of proteins bound to IPd protein
ID protein complexes
Isolation and identification
of protein binding
partners
TAP tag method- identifying
new binding partners
SPR
Surface plasmon resonance
Biomolecular interaction analysis
BIACORE
Protein is immobilized onto surface
Light is refracted onto thin metal
layers
Immobile protein refractive index
changes when ligand is bound
Protein arrays
ELISA based format:
Ab, proteins, peptides immobilized
Solution to be searched is layered on
top
Binding of partner proteins is
detected by SPR or fluorescence
Protein crosslinking
X-linking agent locks interacting
proteins
Formaldehyde
Linking is highly specific
Can be performed in vivo
Cell extract can be subjected to IP
assays
Identify x-linked proteins via co-IP
Chromatin IP ID bound DNA
sequences
Fluorescent protein
tags
Protein-protein interactions with fluorescence
energy transfer (FRET)

Visualizing protein localization

Green fluorescent protein (GFP)
Other ways of exploiting protein
chemistry in research
Singh, Amit et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11346-11351
 Singh, Amit et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11346-11351

Copyright 2006 by the National Academy of Sciences

 Singh, Amit et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11346-11351

Copyright 2006 by the National Academy of Sciences

Singh, Amit et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11346-11351
Singh, Amit et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11346-11351
Singh, Amit et al. (2006) Proc. Natl. Acad. Sci. USA 103, 11346-11351