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BCM5703
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The basic techniques
Concentration (size) Electrophoresis
(size/charge)
precipitation
"native"
ultrafiltration denaturing
dialysis isoelectric focusing
centrifugation 2-dimensional
Chromatography Immunological
(size/charge/chemistry) (size/charge/chemistry)
ion exchange chromatography
size exclusion in situ imaging
affinity immunoblotting
Guidelines for protein purification
Define objectives
Define properties of target protein and
critical contaminants
Minimize the number of steps
Use a different technique at each step
Develop analytical assays
N-terminal sequencing,
antigen for antibody Moderately high < 95%
production, NMR
Separation of proteins based on
physical and chemical properties
Solubility
Binding interactions
Isoelectric Point
Cell growth,
protein over-
expression
Cell lysis
Removal of
cell debris
Why use heterologous expression?
Site-directed mutagenesis
Protein engineering
Expression systems
Bacteria
Escherichia coli
Lactococcus lactis
other bacteria
Yeast
Pichia pastoris
Pichia methanolica
Saccharomyces cerevisiae
Insect cells
baculovirus
Mammalian cells
Cell Free
wheat germ extract
Escherichia coli extract
Generation Time
Time required for cell to divide/for
population to double
Average for bacteria is 1-3 hours
E. coli generation time = 20 min
20 generations (7 hours), 1 cell becomes 1
million cells!
Standard Growth Curve
Phases of Growth
Lag phase making new enzymes in
response to new medium
Log phase exponential growth
Desired for production of products
Most sensitive to drugs and radiation during
this period
Phases of Growth
Stationary phase
nutrients becoming limiting or waste products
becoming toxic
death rate = division rate
Death phase death exceeds division
Expression System Characteristics
Characteristics E. coli Yeast Insect Mammalian
doubling time rapid (30 min) rapid (90 min) slow (18-24 hrs) slow (24 hrs)
protein folding refolding may be refolding may proper folding proper folding
required be required
-carboxylation no no no yes
Translation of
mRNA
No +
MW DinI IPTG IPTG
Target
protein
Protein isolation, concentration, and
stabilization
Reversible
precipitation
with salt or
organic
molecules
Fractional precipitation of proteins
Discard pellet
Precipitate
contaminants
Precipitate Discard
protein of supernatant,
interest Resuspend
protein
Precipitation of proteins
by salting out
_ _ _ _ _ _ _ _
Anions: SCN < ClO4 < NO3 < Br < Cl < acetate < SO4 2 < PO4 3
Liquid
chromatography
(lower resolution,
lower cost)
An introduction to liquid
chromatography
Protein solution applied to a
column
Proteins separated
by chromatography
are collected in
fractions to keep
them separated
Equipment for liquid chromatography
Adsorption Chromatography
Proteins bind to stationary phase
Proteins eluted by altering mobile phase
Includes: affinity, hydrophobic interaction, ion exchange, and
chromatofocusing
Smaller Proteins
Bigger Proteins
Affinity Chromatography
Affinity Chromatography
www.2010.igem.org
Which Tag to Use?
Specificity of binding interaction
Cost of resin
Presence of metals
Tag removal
Tag Removal
considerations:
effect on structure
effect on function
DDDDK flexibility
protease
protein 1 sequence
Tag Removal
Liquid
chromatography
(higher resolution,
higher cost)
Conductivity
Sephacryl S-100
Load in Tris buffer + 200 mM KCl
UV
Elute with Tris buffer + 200 mM KCl
Increasing [Salt]
Increasing Volume and Fraction #
Types of liquid chromatography
Negatively charged pH
Chromatofocusing Adsorption Isoelectric Point Poly-buffer Mono-P
ions gradient
Size Exclusion Solution Size / Shape of Sephacryl #,
Pores Same Buffer
(gel filtration) Phase Protein Sephadex #
Liquid chromatography techniques
advantages and disadvantages
Type of
Advantages Disadvantages Resolution
Chromatography
Resins and ligands
Affinity Quick and specific
can be expensive Low to Medium
Can be used directly Relatively low
Hydrophobic
from ammonium resolution and binding Low to Medium
Interaction sulfate precipitation capacity
pH gradient can be
Chromatofocusing High resolution
harsh for protein High
Distinct from other
techniques, Can be
Size Exclusion used analytically or for
Long run time Low to High
buffer exchange
Protein detection methods
SDS-PAGE
Visual confirmation
UV Spectrophotometry
Absorbance @ 280 nm
Due mostly to Trp
Colorimetric Techniques
Color change proportional to [protein]
Bradford, Lowry, etc
J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29
Electrophoresis
Tris-glycine buffer
10% SDS
Final steps in purification
Check purity by detection methods
Test for interfering contaminants
Nucleases
Proteases
Toxins
Liquid
chromatography
(higher resolution,
higher cost)
Guidelines for protein purification
Define objectives
Define properties of target protein and
critical contaminants
Minimize the number of steps
Use a different technique at each step
Develop analytical assays