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Methods of Protein Purification

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September 9, 2009. Why purify a protein? Characterize function, activity, structure ... Mutant proteins especially difficult to purify ... – PowerPoint PPT presentation

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Title: Methods of Protein Purification


1
Methods of Protein Purification
  • Rachel Britt
  • Cox Lab
  • Biochemistry Addition, Rm 337
  • britt_at_wisc.edu
  • Biochemistry 660
  • September 9, 2009

2
Why purify a protein?
  • Characterize function, activity, structure
  • Use in assays
  • Raise antibodies
  • many other reasons ...

3
Why purify E. coli DinI?
Ala 45
  • Damage Inducible Protein I
  • Recombination mediator
  • Mutant proteins especially difficult to purify
  • New purification strategy discussed throughout
    lecture

Arg 43
Glu 4
Ser 51
4
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

Adapted from Protein Purification Handbook.
Amersham Biosciences. 18-1132-29, Edition AC
5
How pure should my protein be?
6
How pure should DinI be?
7
Separation of proteins based on physical and
chemical properties
  • Solubility
  • Binding interactions
  • Surface-exposed hydrophobic residues
  • Charged surface residues
  • Isoelectric Point
  • Size and shape

8
Basic scheme of protein purification
From Protein Purification Handbook. Amersham
Biosciences. 18-1132-29, Edition AC
9
Protein preparation, extraction, clarification
Cell growth, protein over-expression Cell
lysis Removal of cell debris
10
Why use heterologous expression?
  • Proteins with low natural abundance
  • Proteins predicted by reverse genetics
  • Site-directed mutagenesis
  • Protein engineering

11
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

12
Expression System Characteristics
13
Expression system for DinI?
14
Expression of DinI in E. coli
Plasmid with dinI
DinI expression
transformation
E. coli
15
Inducible promoter system
repressor
promoter
operator
gene to be expressed
induction
promoter
operator
gene to be expressed
RNA Pol
promoter
operator
gene to be expressed
16
Inducible promoter systems
17
Expression of DinI?
18
Expression of DinI
T7 RNA pol
. . . plasmid
plasmid . . .
T7 RNA pol promoter
dinI
Translation of dinI mRNA
No IPTG
IPTG
MW
DinI
DinI !
19
Protein isolation, concentration, and
stabilization
Reversible precipitation with salt or organic
molecules
20
Fractional precipitation of proteins
Discard pellet
Precipitate contaminants
Add Precipitant, Centrifugation, Discard
supernatant, Resuspend protein
Add Precipitant, Centrifugation
Chromatography
Precipitate protein of interest
Discard supernatant, Resuspend protein
21
Precipitation of proteins by salting out
The ability of a salt to precipitate proteins is
described by the Hofmeister series
Anions SCN_ lt ClO4_ lt NO3_ lt Br_ lt Cl_ lt
acetate_ lt SO42_ lt PO43_
Cations Na lt K lt NH4
22
Precipitation of proteins with organic polymers
Adapted from Protein Purification Handbook.
Amersham Biosciences. 18-1132-29, Edition AC
23
Fractional precipitation of DinI?
24
Intermediate Purification
Liquid chromatography (lower resolution, lower
cost)
25
An introduction to liquid chromatography
  • Protein solution applied to a column
  • Column solid porous matrix (stationary phase)
    liquid (mobile phase)
  • Proteins separated based on differing
    interactions with stationary and mobile phases
  • Mobile phase conditions can be adjusted to
    increase or decrease affinity of protein for
    stationary phase (gradient)

26
Fractionation during chromatography
Proteins separated by chromatography are
collected in fractions to keep them separated
27
Equipment for liquid chromatography
ÄKTA FPLC
  • Refrigeration
  • Buffer reservoirs
  • Gradient maker
  • Way to apply buffers and protein sample to column
  • Column
  • Detection system
  • Fraction collector
  • Controller / Recorder

28
Controller / recorder for liquid chromatography
29
Sequence of a general chromatography run
30
Changing buffer between chromatography steps
Dialysis
  • Need porous membrane with specific molecular
    weight cutoff (MWCO)
  • Proteins stay inside membrane
  • Molecules smaller than MWCO free to equilibrate
    across membrane
  • Generally consists of 3, 2-hour steps
  • Rule of thumb volume of solution to change into
    gt 100x volume of protein solution

http//matcmadison.edu/biotech/resources/proteins/
labManual/chapter_4/section4_3.htm
31
Types of liquid chromatography
  • Adsorption Chromatography
  • Proteins bind to stationary phase
  • Proteins eluted by altering mobile phase
  • Includes affinity, hydrophobic interaction, ion
    exchange, and chromatofocusing
  • Solution Phase Chromatography
  • Proteins do not bind to stationary phase
  • Progress of proteins through column impeded by
    matrix of stationary phase
  • Includes size exclusion chromatography (aka gel
    filtration)

32
Size exclusion chromatography
Smaller Proteins
Bigger Proteins
33
Affinity Chromatography
34
Affinity Chromatography
  • Most commonly-used adsorption chromatography
    technique
  • Can be used on protein with natural ligands
  • Often involves covalent attachment of affinity
    tag to protein
  • Because of unique tag, provides rapid, specific
    cleanup in one chromatography step
  • Can allow for automation of protein purification

35
Popular Small Affinity Tags
36
Popular Large Affinity Tags
37
Which Tag to Use?
  • Specificity of binding interaction
  • Cost of resin
  • Native vs. denaturing elution
  • Presence of metals
  • Expression level, solubility toxicity of target
    protein
  • Tag removal

38
Tag Removal
protein
NH2
tag
linker
considerations effect on structure effect on
function flexibility protein 1 sequence
DDDDK
protease
39
Tag Removal
40
Which tag for DinI?
41
Affinity purification of DinI
DinI eluted with gradient of imidazole
Adapted From
42
Affinity purification of DinI
Increasing imidazole
Flowthrough
MW
DinI
DinI !
43
Types of liquid chromatography
44
Liquid chromatography and DinI
45
Anion Exchange and DinI
UV
Q-sepharose column Equilibrate load in Tris
buffer no salt Elute with linear gradient to 1
M KCl
buffer w/ 1M KCl
Conductivity
Increasing KCl in of total buffer
Increasing Abs _at_ 280 nm
Increasing Salt
Increasing Volume and Fraction
46
Anion Exchange and DinI
Fraction Samples
UV
Load
MW
X1
X2
B12
B8
B6
B4
B2
C7
C9
C12
B10
buffer w/ 1M KCl
Conductivity
Pooled Fractions
Increasing KCl in of total buffer
Increasing Abs _at_ 280 nm
Increasing Salt
Increasing Volume and Fraction
47
Polishing steps
Liquid chromatography (higher resolution, higher
cost)
From Protein Purification Handbook. Amersham
Biosciences. 18-1132-29, Edition AC
48
Size Exclusion and DinI
Conductivity
Sephacryl S-100 Load in Tris buffer 200 mM
KCl Elute with Tris buffer 200 mM KCl
UV
buffer w/ 200 mM KCl
Increasing KCl in of total buffer
Increasing Abs _at_ 280 nm
Increasing Salt
Increasing Volume and Fraction
49
Size Exclusion and DinI
Fraction Samples
Conductivity
Load
MW
G3
G4
G5
G6
G8
G9
G10
G11
G12
H12
G7
UV
buffer w/ 200 mM KCl
Pooled Fractions
Increasing KCl in of total buffer
Increasing Abs _at_ 280 nm
Increasing Salt
Increasing Volume and Fraction
50
Liquid chromatography techniques advantages and
disadvantages
51
Protein detection methods
  • SDS-PAGE
  • Visual confirmation
  • UV Spectrophotometry
  • Absorbance _at_ 280 nm
  • Due mostly to Trp
  • Protein calculated with Beers Law
  • Colorimetric Techniques
  • Color change proportional to protein
  • Bradford, Lowry, BCA

J.S.C. Olson and John Markwell. Current
Protocols in Protein Science (2007) 3.4.1-3.4.29
52
UV Spectrophotometry and DinI
DinI e280 1.44 x 104 M-1cm-1 Abs elc C
0.1456 / (1.44 x 104 M-1cm-1 x 1 cm) x 23.33
C 0.000242 M or 242 mM or 9.2 mg/ml
53
Final steps in purification
  • Check purity by detection methods
  • Test for interfering contaminants
  • Nucleases
  • Proteases
  • Toxins
  • Concentrate your protein
  • Precipitation
  • Centricons
  • Small column with high binding capacity
  • Choose a storage buffer and storage conditions
  • Consider intended use of protein
  • Stabilizing additives
  • Flash freeze protein and store at -80o C

54
Basic scheme of protein purification
Liquid chromatography (lower resolution, lower
cost)
Cell growth, protein over-expression Cell
lysis Removal of cell debris
Reversible precipitation with salt or organic
molecules
Liquid chromatography (higher resolution, higher
cost)
55
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

Adapted from Protein Purification Handbook.
Amersham Biosciences. 18-1132-29, Edition AC
56
For your reference . . .
57
Helpful references and guides
  • Amersham Biosciences Protein purification
    handbook. 18-1132-29, Edition AC. Go to
    following URL and download pdf of Protein
    Purification Handbook
  • http//www4.gelifesciences.com/aptrix/upp01077.nsf
    /Content/orderonline_handbooks
  • J.S.C. Olson and John Markwell. Assays for
    Determination of Protein Concentration. Current
    Protocols in Protein Science (2007) 3.4.1-3.4.29
  • http//media.wiley.com/CurrentProtocols/0471111848
    /0471111848-sampleUnit.pdf
  • Alan Williams. Chromatofocusing. Current
    Protocols in Protein Science (1995) 8.5.1-8.5.10
  • http//mrw.interscience.wiley.com/emrw/97804711408
    63/cp/cpps/article/ps0805/current/pdf
  • D.L. Nelson and M.M. Cox. Lehninger Principles of
    Biochemsitry. W.H. Freeman and Co., New York.
    Chapter 3.3 (fourth or fifth edition) (2005 and
    2008 respectively).
  • More in-depth reading Scopes, Robert, K. Protein
    Purification Principles and Practice (Third
    Edition). Springer-Verlag New York, Inc. (1994).
  • Protein Expression Stevens, R.C Structure 8
    (2000) R177-R185.
  • www.genwaybio.com (click on Support/FAQs and
    Answers/Protein Expression)
  • Affinity Purification Arnau, J., Lauritzen, C.,
    Petersen, G.E., Pedersen, J. Prot. Expr. Purif.
    48 (2006) 1-13.

58
Common additives and their uses
Adapted from Protein Purification Handbook.
Amersham Biosciences. 18-1132-29, Edition AC
59
Precipitation with (NH4)2SO42- based on
saturation
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