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Methods of Protein Purification. Rachel Britt Cox Lab Biochemistry Addition, Rm 337 britt@wisc.edu Biochemistry 660 September 9, 2009. Why purify a protein?. Characterize function, activity, structure Use in assays Raise antibodies many other reasons ... . Why purify E. coli DinI?.

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methods of protein purification

Methods of Protein Purification

Rachel Britt

Cox Lab

Biochemistry Addition, Rm 337

britt@wisc.edu

Biochemistry 660

September 9, 2009

why purify a protein
Why purify a protein?
  • Characterize function, activity, structure
  • Use in assays
  • Raise antibodies
  • many other reasons ...
why purify e coli dini
Why purify E. coli DinI?
  • Damage Inducible Protein I
  • Recombination mediator
  • Mutant proteins especially difficult to purify
  • New purification strategy discussed throughout lecture

Ala 45

Arg 43

Glu 4

Ser 51

guidelines for protein purification
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

separation of proteins based on physical and chemical properties
Separation of proteins based on physical and chemical properties
  • Solubility
  • Binding interactions
  • Surface-exposed hydrophobic residues
  • Charged surface residues
  • Isoelectric Point
  • Size and shape
basic scheme of protein purification
Basic scheme of protein purification

From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

protein preparation extraction clarification
Protein preparation, extraction, clarification

Cell growth, protein over-expression

Cell lysis

Removal of cell debris

why use heterologous expression
Why use heterologous expression?
  • Proteins with low natural abundance
  • Proteins predicted by reverse genetics
  • Site-directed mutagenesis
  • Protein engineering
expression systems
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
expression of dini in e coli
Expression of DinI in E. coli

Plasmid with dinI

DinI

expression

transformation

E. coli

inducible promoter system
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

expression of dini18
Expression of DinI

T7RNA pol

. . . plasmid

plasmid . . .

T7 RNA pol promoter

dinI

Translation of dinI mRNA

No IPTG

+ IPTG

MW

DinI

DinI !

protein isolation concentration and stabilization
Protein isolation, concentration, and stabilization

Reversible precipitation with salt or organic molecules

fractional precipitation of proteins
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

precipitation of proteins by salting out
Precipitation of proteins by “salting out”

The ability of a salt to precipitate proteins is described by the Hofmeister series:

Anions: SCN_ < ClO4_ < NO3_ < Br_ < Cl_ < acetate_ < SO42_ < PO43_

Cations: Na+ < K+ < NH4+

precipitation of proteins with organic polymers
Precipitation of proteins with organic polymers

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

intermediate purification
Intermediate Purification

Liquid chromatography (lower resolution, lower cost)

an introduction to liquid chromatography
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)
fractionation during chromatography
Fractionation during chromatography

Proteins separated by chromatography are collected in fractions to keep them separated

equipment for liquid chromatography
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
changing buffer between chromatography steps dialysis
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 > 100x volume of protein solution

http://matcmadison.edu/biotech/resources/proteins/labManual/chapter_4/section4_3.htm

types of liquid chromatography
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)
size exclusion chromatography
Size exclusion chromatography

Smaller Proteins

Bigger Proteins

affinity chromatography
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
which tag to use
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
tag removal
Tag Removal

protein

NH2–

tag

linker

considerations:

effect on structure

effect on function

flexibility

protein 1° sequence

DDDDK

protease

affinity purification of dini

DinI

Nickel

Affinity purification of DinI

DinI eluted with gradient of imidazole

Adapted From:

affinity purification of dini42
Affinity purification of DinI

Increasing [imidazole]

Flowthrough

MW

DinI

DinI !

slide45

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 @ 280 nm

Increasing [Salt]

Increasing Volume and Fraction #

slide46

Anion Exchange and DinI

Fraction Samples

UV

Load

MW

X1

X2

B12

B8

B10

B6

B4

B2

C7

C9

C12

% buffer w/ 1M KCl

Conductivity

Pooled Fractions

Increasing [KCl] in % of total buffer

Increasing Abs @ 280 nm

Increasing [Salt]

Increasing Volume and Fraction #

polishing steps
Polishing steps

Liquid chromatography (higher resolution, higher cost)

From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

slide48

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 @ 280 nm

Increasing [Salt]

Increasing Volume and Fraction #

slide49

Size Exclusion and DinI

Fraction Samples

Conductivity

Load

MW

G3

G4

G5

G6

G7

G8

G9

G10

G11

G12

H12

UV

% buffer w/ 200 mM KCl

Pooled Fractions

Increasing [KCl] in % of total buffer

Increasing Abs @ 280 nm

Increasing [Salt]

Increasing Volume and Fraction #

protein detection methods
Protein detection methods
  • SDS-PAGE
    • Visual confirmation
  • UV Spectrophotometry
    • Absorbance @ 280 nm
    • Due mostly to Trp
    • [Protein] calculated with Beer’s 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

uv spectrophotometry and dini
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

final steps in purification
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
  • Confirm identity of purified protein
    • Mass spectrometry
    • N-terminal sequencing
    • Analytical assays
basic scheme of protein purification54
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)

guidelines for protein purification55
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

helpful references and guides
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/9780471140863/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.
  • Affinity Purification: Lichty, J.J. et al. Prot. Expr. Purif. 41 (2005) 98-105.
  • Affinity Purification: Waugh, D.S. TRENDS Biotech. 23 (2005) 316-320.
common additives and their uses
Common additives and their uses

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC