protein expression and purification
TRANSCRIPT
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Protein Expression and Purification
James Chou
BCMP201 Spring 2008
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The lac repressor inhibits the expressionof genes coding for proteins involved inthe metabolism of lactose in bacteria; it isinactivated by IPTG.
lac operon
Bacterial expression system
T7 promoter lac operator
T7 RNA polymerase
lac repressor (inactivated by IPTG)
protein DNA sequence Terminator
Amp r
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Bacterial Strains for Protein Expression
Bacterial Strain
Features Usage
BL21(DE3) (Novagen)
defficient in lon and omp-t proteas e s Non-toxic proteins
BL21(DE3)-pLysS (Novagen)
reduces basal level expression of the gene of intere s t
More toxic proteins
C41(DE3) (Lucigen)
prevents cell death associated with expression of many toxic protein s
Toxic membrane proteins
C43(DE3) (Lucigen)
Effective in expressing toxic and membrane proteins from all classes of organisms, including viruses, eubacteria, archaea, yeasts, plants, insects, and mamma l s
Toxic, difficult proteins
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The Luria Broth (LB) rich medium
10 g tryptone (peptide digested from casein by trypsin)
5 g yeast extract
5 g NaCl
in 1 litre with distilled water
Cell growth
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The minimal (M9) medium
Supplement with 1 tabletCentrum A to Zinc
6.8 g Na2HPO4
3.0 g KH2PO4
1.0 g NH4Cl
0.5 g NaCl
4.0 g Glucose
2 mM MgSO4
0.1 mM CaCl2
pH ~ 7.2
sole source of N,use 15NH4Cl forNMR studies
sole source of C, use13C-glucoes for NMRstudies
Components for 1 L
1mM of antibiotics, e.g., ampicillin, kanamycin
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The standard cell growth conditions
37 °C
shake ~300 rpm
Constant feed of O2, NH4OH,glucose
Can reach OD600 ~ 50 !!
OD600 limited by O2
flask volume > 4 xmedium
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A typical cell growth curve
lag phase
log phase
stationaryphase
declinephase
IPTGinduction
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IMPORTANT: always screen for expression conditions
protein
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Example of expressionvector - His-tag(Novagen)
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Ni NTA resin
Lyse cell
Wash with buffer containing ~10mM imidazole
Elute protein with 300 mM imidazole
Imidazole, competitive binderof His tag
Spin the lysate at 15,000 g for 30 min
Load supernatant to Ni-NTA column
General protocol for purification of His-tagged proteins
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Further purify by size exclusion
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Example of expression vector - GST fusion
GST (26 kDa) Protein of interest
protease site
pGEX-2TK Thrombin (37 kDa)
pGEX-5X-1 Factor Xa (48 kDa)
pGEX-6P-1 Precision protease (46 kDa)
Vectors from Amershan
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Glutathione S-transferase (GST)
Glutathione
Glutathione affinity purification
Glutathione Sepharose
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Amersham
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Further purify by size exclusion
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Example of expression vector - MBP fusion
MBP (45 kDa)
Tobacco etch virus (TEV) protease cleavage site
Protein of interest
maltose
Maltose Binding Protein (MBP)
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Purification of MBP-fusion by amylose/dextrin column
amylose
SepharoseTM High Performance based on a34 µm bead-sized matrix.
dextrin
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Introduction to Chromatography
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Plate Theory - Martin & Synge 1954
View column as divided into a number (N) of adjacent imaginarysegments called theoretical plates within each theoreticalplate complete equilibration of analytes between stationaryand mobile phase occurs
The Theory of Chromatography
Mobile Phase ! "!# !! Stationary Phase
Sampleinjection
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Elution profile is a Gaussian function when N is large
dX
Stationary= K dX
Mobile
Predicting elution profile using the plate theory
flow direction
mobile V
M
X
M p( )
mobile V
M
X
M p!1( )
mobile V
M
X
M p+1( )
stationary V
S
X
S p( )
stationary V
S
X
S p-1( )
stationary V
S
X
S p+1( )
V
i= volume of phase i
X
i= analyte conc. in phase i
! mass( ) = X
M p-1( )" X
M p+1( )( )!V
!V = volume of solution from
one plate to the next
. . .
N = number of plates
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Bandwidth of the elution profile
tR = retension time of protein, the time between sample injection andan analyte peak reaching a detector at the end of the column
tM = retension time of the mobile phase (for reference)
N = 5.55 tr2 / w1/22 , w1/2 is width of the elution peak at half-height
time
w
1 2
tR
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Greater separation occurs :–with greater number of theoretical plates (N)–as plate thickness becomes smaller
N = 5.55 tr2 / w1/22
Predictions from the Plate Theory
The width of bands increases as their retentionvolume increases
The thinner the plate, the narrower the eluted peak
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Column Dimensions
Nmax = 0.4 * L / dp
N - maximum column efficiency (# of plates)L - column lengthdp - particle size
Column length L = n x thickness of the plates
So, the smaller the particle size the higher the efficiency!
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Ion exchange chromatography
++ +
+++
-
- -
-
--
++ +
+++
-
-
--
--
++ +
+++
-
- -
--
+
-
-+
+
- -
-+ +
-
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Common functional groups used on ion exchangers
strongweak
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Common resin material
Sepharose -- crosslinked, beaded-form of agarose
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Sepharose ion exchangers
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MonoBeads Exchanger
10 μm beaded hydrophilic polystyrene/divinyl benzene resin,pH stable, porous
Mono S - substituted with methyl sulphonate groups to yield thestrong cation exchanger
Mono Q - substituted with quaternary amine groups to yield thestrong anion exchanger
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pI of the protein and pH of the mobile phase is important
Example
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Size exclusion chromatography
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Larger molecules move faster through the column
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Example: desalting of a protein sample
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Optimize resolution
Factors affecting resolution
- ratio of starting sample volume to the column volume
- flow rate
- column dimension
- particle size, pore size, packing density
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Smaller sample volume -> better separation
Results from the Superdex 200 HR 10/30 column
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RS=
Vr1!V
r2
w1+ w
2( ) 2
Longer column increases resolution
Resolution in chromatography
RS increases as column bed height
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Faster flow rate decreases resolution
Results from Amershan HiLoad 16/60 Superdex 30 column
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Commonly used columns in protein purification
Amersham HiLoad Superdex 75/200 prep column
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Hydrophobic interaction between polypeptide and the stationary phase
Reversed phase chromatography (native)
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Modify hydrophobic interactions with salts
Hofmeister series: the effect of anions and cations on proteinprecipitation
Relative effects of some salts on protein precipitation.
Increase hydrophobic interaction
decrease hydrophobic interaction
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Reversed phase chromatography (denatured)
Advantages
- resolution (pure) - can purify anything
Disadvantages
- harsh solvent condition - denature protein
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Reversed phase HPLC columns are packed with silica beads
- Si - OH
- Si - OH
Pore size (affect binding capacity)100 A for small peptide
300 A for proteins
Bead size
3 - 5 um for small prep (slow flow)
> 15 um for large prep (fast flow)
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Modify hydrophobicity of the column by varying thehydrocarbon length
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The lower the polarity of the mobile phase, the greater itseluting strength. The non-polar organic solvents are used toelute proteins.
The most widely used organic solvents (no UV absorption)
- acetonitrile - methanol - isopropanol (viscous, high back pressure)
Properties of the mobile phase for eluting the protein
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The retention of polypeptides can be modified by mobilephase pH due to ionizable groups in the peptide. - suppress the ionization the acidic groups in protein
The most widely used acid - trifluoroacetic acid (TFA) - ortho-phosphoric acid
Strong acids are also used in reversed phase HPLC
Denature the protein at ~ pH 2-3 to expose all hydrophobicgroups.
Base is normally not used because the silica matrix beaksdown above pH 8.
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Example of reversed phase HPLC