Protein Purification

1
Protein Purification

• Tutorial
• Minnie Murugesan
• Dr. Scotts Group
• 07-12-05

2
Eventful Protein Purification

• Grow cells in media (vectortag)
• Centrifuge, Collect the pellet
• Lyse the cells (appropriate buffer)

Pilot Expression SDS PAGE, Assay
Purification Strategy
Characterization Mass Spectroscopy X-ray
Crystallography Functional Assay
Solubility Aggregation Recombination
3
OUTLINE

• Chromatography techniques
• Affinity Chromatography (AC)
• Hydrophobic Interaction Chromatography (HIC)
• Ion Exchange Chromatography (IEC)
• Gel Filtration (GF)
• Capillary electrochromatography (CEC)
• Other New Strategies for Protein Purification
• Solubility, Aggregation and Re-folding of
  Proteins

Invented by a Russian botanist named Mikhail
Tswett in 1903. He separated plant pigments using
glass columns packed with calcium carbonate.
4
Protein Purification Strategies

• Evaluate an assay for the protein of interest
• Shortlist a method to have a reasonable source
  for that activity

(http//www5.amershambiosciences.com/aptrix/upp009
19.nsf/Content/LabSep_EduC5CAboutPurBiom5CHowToC
ombine)
5
Three Phase Strategy
(www.amershambiosciences.com)
6
Chromatographic Modes of Protein Purification
(Christian G. Huber, Biopolymer Chromatography,
Encylcopedia in analytical chemistry, 2000)
7
Affinity Chromatography
Affinity Chromatography Surface bound with Epoxy,
aldehyde or aryl ester groups
Metal Interaction Chromatography Surface bound
with Iminodiacetic acid Ni2/Zn2/Co2
(Christian G. Huber, Biopolymer Chromatography,
Encylcopedia in analytical chemistry, 2000)
8
Metal Interaction Chromatography (AC)

• Points to Note
• Avoid chelating agents
• Increasing incubation time
• 3. Slow gradient elution

(www.qiagen.com)
9
Affinity Chromatography
Binding Capacity (mg/ml) medium 12mg of histag
proteins (MW 27kDa) Depends on Molecular weight
Degree of substitution /ml medium 15mmol Ni2
Backpressure 43psi Change the guard column filter
(Christian G. Huber, Biopolymer Chromatography,
Encylcopedia in analytical chemistry, 2000)
10
Hydrophobic Interaction Chromatography
Biopolymer (phenyl agarose - Binding
Surface) Driving force for hydrophobic
adsorption Water molecules surround the analyte
and the binding surface. When a hydrophobic
region of a biopolymer binds to the surface of a
mildly hydrophobic stationary phase, hydrophilic
water molecules are effectively released from the
surrounding hydrophobic areas causing a
thermodynamically favorable change in
entropy. Temperature plays a strong
role Ammonium sulfate, by virtue of its good
salting-out properties and high solubility in
water is used as an eluting buffer
(Christian G. Huber, Biopolymer Chromatography,
Encylcopedia in analytical chemistry, 2000)
11
Ion Exchange Chromatography
Fractogel matrix is a methacrylate resin upon
which polyelectrolyte Chains (or tentacles) have
been grafted. (Novagen)
(www.novagen.com)
12
Gel Filtration
(http//lsvl.la.asu.edu/resources/mamajis/chromato
graphy/chromatography.html)
13
Immuno Affinity Chromatography
(http//www.cellmigration.org/resource/discovery/d
iscovery_proteomics_approaches.html)
14
DNA Binding Proteins

ATP immobilized on polyacrylamide resin
Heparin Sepharose Negatively charged proteins (pI
gt7) are not captured/separated effectively.
(www.novagen.com)
15
Capillary Electrochromatography

• CEC is an electrokinetic separation technique
• Fused-silica capillaries packed with stationary
  phase
• Separation based on electroosmotically driven
  flow
• Higher selectivity due to the combination of
  chromatography and electrophoresis
• Fused silica tube filled with porous
  methacrylamide-stearyl methacrylate-dimethyldially
  l ammonium chloride monolithic polymers, 80 x
  0.5mm i.d., 5.5kV. High Plate count 400,000

Height Equivalent to a Theoretical Plate /Plate
Count (HETP) H L/N number of plates N
16(t/W)2 where L column length, t retention
time, and W peak width at baseline
(http//www.capital-hplc.co.uk)
16
CEC columns
AC, IEC columns
CEC column
NP, RP columns
17
Commercially available protein purification kits
GSTBind Purification Kits HisBind
Purification Kits Magnetight Oligo d(T)
Beads MagPrep Streptavidin Beads Protein A and
Protein G Plus Agaroses STag Purification
Kits Streptavidin Agarose T7Tag Affinity
Purification Kit ProteoSpin CBED (Concentration,
Buffer Exchange and Desalting) Maxi Kit
Effectively desalts and concentrates up to 8 mg
of protein with an efficient, easy-to-use
protocol.(Norgen Biotek Corporation) ProteoSpin
Detergent Clean-up Micro Kit Provides a fast
and effective procedure to remove detergents
including SDS, Triton X-100, CHAPS, NP-40 and
Tween 20.
(http//www.emdbiosciences.com)
18
Schematic of a Multi-dimensional Separation System
19
Fast Protein Liquid Chromatograph (FPLC)
(www.pharmacia.com)
20
Protein Analysis
(http//www.cellmigration.org/resource/discovery/d
iscovery_proteomics_approaches.html)
21
Detection of Proteins by Derivatization with
Higher Sensitivity
1000 times more sensitive than UV-Vis detection
(Christian G. Huber, Encylcopedia in analytical
chemistry, 2000)
22
Solubility of a protein

• Depends strongly on the composition of the lysis
  buffer.
• Salt concentration
• Freeze-thaw protocol
• Freeze quickly on dry ice and leave for 3
  min.
• Thaw immediately at 42 C. Vortex vigorously
  to mix well.
• Repeat the two previous steps three more
  times (4 cycles in all).

• Membrane proteins
• 1. Removal of unbroken cells from the cell lysate
  by low speed centrifugation (20 min at 10,000 g).
  
• 2. Isolation of the membrane particles from the
  supernatant by ultracentrifugation (60 min at
  gt100 000 g).
• 3. Washing of the membrane particle to remove all
  soluble proteins.
• Solubilization of protein from the membrane
  particles by a mild detergent. (detergent
  protein ratio 110)
• 5. Phosphate buffers(0.1M-0.5M), 5-50 glycerol
  helps.

(http//www.ls.huji.ac.il/purification)
23
Protein Aggregation

• Numerous physicochemical stresses can induce
  protein aggregation
• Heat, pressure, pH, agitation, freeze-thawing,
  dehydration, heavy metals,
• phenolic compounds, and denaturants.

(http//www.integritybio.com/protein20aggregation
.htm)
24
Solubilization of Aggregated Proteins
Denaturation and Renaturation Variables Good
starting point Buffer composition (pH, ionic
strength) 50 mM Tris-HCl, pH 7.5 Incubation
temperature 30C Incubation time 60
min Concentration of solubilzing agent 6 M
guanidine-HCl or 8 M urea Total protein
concentration 1-2 mg/ml
Re-folding of Proteins The addition of a mixture
of reduced and oxidized forms of low molecular
weight thiol reagent usually provides the
appropriate redox potential to allow formation
and reshuffling of disulfide bonds (1-3 mM
reduced thiol and a 51 to 11 ratio of reduced
to oxidixed thiol) The most commonly used are
glutathione, cysteine and cysteamine.
(www.biovectra.com)
25
Reagents used for Re-folding of proteins
(http//www.ls.huji.ac.il/purification)
26
Reagents used for Re-folding of proteins
(Continued)
(http//www.ls.huji.ac.il/purification)
27
6xHis Tagged Protein Detection Directly on the
Gel (from Pierce) E. coli lysates expressing
6xHis-tagged proteins, stained with the Pierce
6xHis Protein Tag Staining Kit 
(www.piercenet.com)
28

• University of Oklahoma
• Recombinant Protein Solubility Prediction
• Type (or cut and paste) your protein sequence
  below, click on the "Submit" button, and the
  solubility probability of your protein will be
  calculated.
• The statistical model predicts protein
  solubility assuming the protein is being
  over-expressed in Escherichia coli.
• The input protein sequence has a 73.4 percent
  chance of insolubility when overexpressed in E.
  coli. - mbh8 NADH dehydrogenase subunit -
  integral membrane protein (NP_579159)
• The input protein sequence has a 80.5 percent
  chance of solubility when overexpressed in E.
  coli. - replication factor A related protein
  (NP_579749)

(www.biotech.ou.edu)