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Protein Purification: From industrial enzymes to
cancer therapy
2
Protein
Expression
and
Purification
Series
Jim DeKloe
Solano Community College
[email protected]
Instructors
Leigh Brown, M.A. (Central US)
[email protected]
Bio-Rad Curriculum and Training Specialists:
Sherri Andrews, Ph.D. (Eastern US)
[email protected]
Damon Tighe (Western US)
[email protected]
3
Protein
Expression
and
Purification
Series
Workshop
Timeline
• Introduction
• Recombinant protein expression and purification for
biomanufacturing
• Dihydrofolate reductase
• Perform affinity chromatography
• Perform size exclusion (desalting) chromatography
• Quantify protein concentration
• Look at SDS-PAGE results
• Look at enzyme results
• Scaling up for the BioLogic LP
4
Protein
Expression
and
Purification
Series
DHFR
Enzymatic
Assay
Module
SDS-PAGE
Electrophoresis
Module
Growth and
Expression
Module
Purification
Module
Option 3
Prepacked
Cartridge
Purification
Module
5
Option 1
Centrifugation
Purification
Module
Option 2
Handpacked
Column
Purification
Module
Why Teach
about Protein
Expression and
Purification?
• Powerful teaching tool
• Real-world connections
• Link to careers and industry
• Tangible results
• Laboratory extensions
• Interdisciplinary – connects
biochemistry, biomanufacturing,
chemistry, biology and medical
science
• Mimics a complete workflow utilized
in research and industry
6
Protein
Expression
and
Purification
Series
Advantages
• Follows a complete workflow including
bacterial cell culture, induction, fractionation,
purification, and analysis of purified protein
• Teaches affinity purification
• Work with a non-colored protein that is
comparable to real world applications
• Includes ability to run at small scale using a
16k microcentrifuge or scaling up and using
chromatography instrumentation
• Possibility of extensions including western
blots, ELISAs, site-directed mutagenesis
studies, induction experiments
7
The Value
of Proteins
Price Per Gram
Bovine Growth Hormone
$14
Gold*
$56
Insulin
$60
Human Growth Hormone
$227,000
Granulocyte Colony
Stimulating Factor
$1,357,000
Prices in 2011 US Dollars
* As of 8/14/2011
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Protein – The product
of Biotech
PROTEIN:
USED IN THE
TREATMENT OF:
Cell
Production
Insulin
Human growth hormone
Granulocyte colony stimulating factor
Erythropoietin
Tissue plasminogen activator
Hepatitis B virus vaccine
Human papillomavirus vaccine
Diabetes
Growth disorders
Cancers
Anemia
Heart attack
Vaccination
Vaccination
E. coli
E. coli
E. Coli
CHO cells
CHO cells
Yeast
Yeast
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Biomanufacturing
Defined
The production of
pharmaceutical proteins using
genetically engineered cells
10
Expression
Choices
Cell type:
• E. coli
• Yeast
• Mammalian
–CHO
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Expression Choices
Parameter
Bacteria
Yeast
Mammalian
Contamination risk
Low
Low
High
Cost of growth
medium
Low
Low
High
Product titer
(concentration)
High
High
Low
Folding
Sometimes
Probably
Yes
Glycosylation
No
Yes, but different pattern
Full
Relative ease to grow
Easy
Easy
Difficult
Relative ease of
recovery
Deposition of product
Difficult
Easy
Easy
Intracellular
Intracellular or extracellular
Extracellular
Product
Intracellular
Often secreted into media
Secreted
12
DHFR —
Dihydrofolate
reductase
•Converts dihydrofolate into tetrahydrofolate
(THF) by the addition of a hydride from
NADPH
•THF is a methyl (CH3) group shuttle required
for synthesis of essential molecules
- nucleotides
- amino acids
13
DHFR and
Cancer
•DHFR inhibition or reduction disrupts nucleic acid
synthesis affecting
-Cell growth
-Proliferation
•Methotrexate – chemotherapeutic agent
-Competitive inhibitor of DHFR
-Methotrexate resistance - correlates with
amplification of DHFR genes
14
Induction
Biotech companies genetically
engineer plasmids to place
genes behind inducible
promoters
15
Transcriptional
Regulation in
the pDHFR
system
lac Operon
LacI
Z
Y A
Effector (Lactose)
LacI
Z
Y A
RNA Polymerase
Z
Lactose
16
Y A
IPTG
Transcriptional
Regulation in
the pDHFR
system
17
Transcriptional
Regulation in
the pDHFR
system
18
Lactose = Induced System
GST-DHFRHis
Construct
GST – DHFR - His
Glutathione-s-transferase
•Added to increase solubility
•Can be used as a secondary
purification methodology
Histidine tag
•6 Histidine tag that binds to
certain metals such as nickel
Human dihydrofolate reductase
•Gene product of interest
19
•Target for chemotherapy reagents
Selection
Mechanism
for
Mammalian
cells
20
Phases of
growth
21
Recovery
Separation of protein from
other molecules
Purification
Separation of the protein of
interest from other proteins
22
Chromatography
Basics
• Mobile phase (solvent and the molecules to
be separated)
• Stationary phase (through which the mobile
phase travels)
– paper (in paper chromatography)
– glass, resin, or ceramic beads (in column
chromatography)
• Molecules travel through the stationary
phase at different rates because of their
chemistry.
23
Types of Column
Chromatography
•Ion Exchange (protein charge)
•Size Exclusion (separates on size)
•Hydrophobic Interaction (hydrophobicity)
•Affinity:
•Protein A  tail of Antibodies
•His-tagged metal complexes (Ni)
•Glutathione-s-transferase  glutathione
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Performing the
chromatographic
separation
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•Gravity Chromatography
•Spin Column Chromatography
•Chromatography Instrumentation
•Small scale
•Biomanufacturing scale
(bioreactors)
Protein
Expression
and
Purification
Series
Workflow
Streak Cells
Overnight culture
Subculture, monitor, and induce
Harvest and lyse cells
Purify
Centrifugation or Instrumentation
Analyze
26
Centrifuge
RCF to RPM
conversion
• Accurate RCF(g) is important for
chromatography resins
• RPM to RCF varies for different models of
centrifuges due to variation in rotor radius
RCF = relative centrifugal force
RPM = rotations per minute
R = radius in cm from center of
rotor to middle of spin column
• Determine RPM for 1,000 x g. The Bio-Rad 16K
microcentrifuge rotor has a radius of 7.3 cm
3,497
1,000
7.3
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Affinity
purification
1.
Pour column
2.
Wash resin to
remove packing
buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
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Elute GST-DHFR-His
A.
Label column with
initials. Snap off bottom
tab of column, remove
cap and place in 2 ml
microcentrifuge tube.
200 µl
B.
C.
Add 200 µl of Ni-IMAC
resin slurry to empty
column
Centrifuge for 2 minutes
at 1,000 x g. After spin,
discard buffer that has
collected in the
microcentrifuge tube.
Ni-IMAC
resin
slurry
discard
Affinity
purification
1.
Pour column
2.
Wash resin to
remove packing
buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFR-His
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200 µl
A.
B.
Add 200 µl of
distilled H2O to
column
Centrifuge for 2 minutes
at 1,000 x g. After spin,
discard water from
collection tube.
Distilled
H2O
discard
Affinity
purification
500 µl
A.
1.
Pour column
2.
Wash resin to remove
packing buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFR-His
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B.
Add 500 µl of
Equilibration buffer to
column
Centrifuge for 2 minutes
at 1,000 x g. After spin,
discard Equilibration
buffer and collection
tube. The column is now
ready to use.
Equilibration
buffer
discard
Affinity
purification
1.
Pour column
2.
Wash resin to remove
packing buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFR-His
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600 µl
A.
Place yellow tip closure on
bottom of column. Add
600 µl Soluble Fraction
to Column; Put on clear
top cap.
B.
Gently mix for 20 min.
Soluble fraction
His tags
Histidine
-OOC
• His tags are typically a series of 6 histidines
added to the C or N terminus of a recombinant
protein
• His tag and column interaction
N3H+
Ni
Resin
His-tagged
Recombinant
Protein
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His tags
• His and imidazole structure similarities
• Imidazole competes with His for Ni2+ sites
Histidine
-OOC
33
N3H+
Imidazole
Affinity
purification
A.
1.
Pour column
2.
Wash resin to remove
packing buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFR-His
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Label three 2 ml tubes:
“Flow through”
“Wash”
“ Eluate”
Affinity
purification
1.
Pour column
2.
Wash resin to remove
packing buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFR-His
B.
Remove yellow tip closure.
C.
Place column in 2 ml collection
tube labeled “Flow Through” and
remove clear top cap.
D.
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Centrifuge for 2 min at 1,000x g.
Set aside Flow Through.
“Flow through”
Keep
“Flow through”
Affinity
purification
1.
Pour column
2.
Wash resin to remove
packing buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFR-His
36
A.
Place column in 2 ml collection
tube labeled “Wash”.
“Wash”
600 µl
B.
Add 600 µl Wash Buffer to
column.
•
Centrifuge for 2 min at 1,000xg.
Set aside Wash fraction.
Wash
Buffer
Keep
“Wash”
Affinity
purification
1.
Pour column
2.
Wash resin to remove
packing buffer
3.
Equilibrate resin
4.
Bind GST-DHFR-His
5.
Elute unbound
proteins
6.
Wash protein bound
onto the resin
7.
Elute GST-DHFRHis
37
A.
Place column in 2 ml collection
tube labeled “Eluate”.
“Eluate”
400 µl
B.
Add 400 µl Elution Buffer to
column.
•
Centrifuge for 2 min at 1,000xg.
Set aside Eluate.
Elution
Buffer
Keep
“Eluate”
Recap so
far….
Soluble
fraction
Started with a complex mixture of all the
soluble E. coli proteins along with the
induced expressed human GST-DHFRHis
Purified the GST-DHFR-His away from the
E. coli proteins by using the affinity of
the 6 Histidine tag on GST-DHFR-His for
Ni-IMAC beads
38
Flowthrough
Wash
~600 µl
~600 µl
Eluate
~400 µl
Size
exclusion
purification
(buffer
exchange)
Eluate
fraction
GST-DHFR-His in 20 mM sodium
phosphate, 300 mM NaCl and
250 mM imidazole
Imidazole
250 mM
imidazole
solution has
an A280= 0.2-0.4
39
W and Y contribute to
A280 of proteins
NEED TO REMOVE IMIDAZOLE TO QUANTIFY
PROTEIN CONCENTRATION USING A280
Principles of
Size Exclusion
Chromatography
• Beads in column are made of polyacrylamide and have tiny pores
• The mixture of molecules is added to the column
• Large molecules move through the column quickly traveling around
the beads
• Smaller molecules move through the pores of the beads and take
longer to pass through the column
40
http://tainano.com/Molecular%20Biology%20Glossary.files/image047.gif
Principles of
Size Exclusion
Chromatography
• The mass of beads in the column is called
the column bed
• Beads trap or sieve and filter molecules
based on size
• The separation of molecules is called
fractionation
• Size of pores in beads determines the
exclusion limit (what goes through the beads and
what goes around the beads)
• Molecules are dissolved in a buffer
41
Size
Exclusion
42
Size
exclusion
purification
(desalting)
1.
Prepare SEC column
2.
Desalt GST-DHFR-HIS
with SEC column
43
A.
Label desalting column with your
initials.
B.
Invert column several times to
resuspend gel.
C.
Snap off bottom tip and place in a
2 ml collection tube.
Size
exclusion
purification
(desalting)
1.
Prepare SEC column
2.
Desalt GST-DHFR-HIS
with SEC column
1.
D.
Remove green top cap and
allow excess packing buffer to
drain by gravity to top of resin
bed. If the column does not
begin to flow, push the cap
back on the column and then
remove to start the flow.
discard
After draining, place column
in clean 2 ml tube.
discard
E.
44
Centrifuge for 2 min at 1,000
x g. Discard 2ml tube
containing packing buffer.
Size
exclusion
purification
(desalting)
1.
Prepare SEC column
2.
Desalt GST-DHFR-HIS
with SEC column
Removing the 250 mM imidazole solution by size
exclusion chromatography
A.
Label new 2 ml tube
“Desalted Eluate”.
B.
Carefully apply 75 ul of
eluate fraction directly to
the center of column. Be
careful not to touch resin
with pipet tip.
75 µl
“Eluate”
2x
45
C.
Centrifuge for 4 min at
1,000 x g.
D.
Repeat addition of 75 µl of
Eluate fraction to column
and centrifugation.
Size
exclusion
purification
(desalting)
Desalted eluate ~150 µl
1.
Prepare SEC column
2.
Desalt GST-DHFR-HIS
with SEC column
GST-DHFR-His
in 10 mM Tris buffer
250 mM Imidazole has been removed
46
Protein
Analysis
47
•
Determination of success of induction, lysis,
and purification of GST-DHFR-His using SDSPAGE analysis
•
Measurement of concentration using the
absorbance at 280 nm
•
Enzymatic activity analysis
1.
Prepare Samples
2.
Prepare TGX Gel and
vertical
Electrophoresis
apparatus
3.
4.
Stain gel
5.
Analyze gel
48
250
150
100
75
Load and Run Gel
50
37
25
20
15
10
9 – Desalted GST-DHFR-His
8 – Eluted GST-DHFR-His
7 – Column wash
6 – Column flow through
5 – Soluble fraction
4 – Insoluble fraction
3 – Induced cells
2 – Uninduced cells
1 – Precision Plus Dual Color
standards
Protein
analysis
SDS-PAGE
Protein
analysis
Quantitation of Protein in Desalted Fraction
(Quantitation
using A280)
Turn on spectrophotometer and set
absorbance to 280 nm. Add 100 µl
distilled H2O to clean UV compatible
cuvette.
Blank spectrophotometer with distilled H2O.
Pipet 100 µl of your desalted eluate sample
(GST-DHFR-His) into clean UV compatible
cuvette.
Measure absorbance of sample at
280nm and record or print the value.
Return sample to 2 ml tube.
49
+100 µl
Clean UV cuvette
Distilled H2O
+ 100 µl
Desalted
eluate
Clean UV cuvette
Protein
analysis
(Quantitation
using A280)
Calculate concentration of GST-DHFR-His
Beer’s Law
A=ecl
e - the molar absorptivity
((mol/L)-1 cm-1)
l - the path length of the sample (usually 1cmcuvette)
C - the concentration of the compound in
solution (mol/L)
Expected results
1.3 x 10-6 – 5.3 x 10-6 M
For GST-DHFR-His
e = 75,540 (mol/L)-1 cm-1
C (mol/L) =
Absorbance
75,540 (mol/L)-1 cm-1 x 1 cm
50
Enzyme
Assay
Absorbance at 340nm
51
Enzyme
Assay
A.
Set up spectrophotometer for
kinetics measurements at 340 nm.
B.
Blanking the instrument. Add 985 µl
1x PBS to cuvette; place in
instrument, read as blank. Save
cuvette with PBS
+ 985 µl
1x PBS
52
C.
Running the no substrate control
reaction. Add 6 µl of 10 mM NADPH
to cuvette containing 985 µl 1x PBS.
Add 15 µl of purified, desalted GSTDHFR-His eluate to cuvette. Cover
cuvette with parafilm and invert 10
times. Immediately place cuvette in
spectrophotometer and begin kinetics
run.
D.
As run is proceeding, record
absorbance value every 15 seconds
for 150 seconds. Remove and save
cuvette from the spectrophotometer.
+ 985 µl
1x PBS
+ 6 µl
NADPH
+ 15ul
desalted
Eluate
Enzyme
Assay
53
E.
Running the enzymatic reaction with the
GST-DHFR-His, NADPH (cofactor) and DHF
(substrate).Note: The enzyme reaction should
be prepared while standing at the
spectrophotometer. The reaction occurs
extremely quickly and it is necessary to place
the cuvette in the spectrophotometer and start
the readings as quickly as possible once the
DHF has been added.
F.
Add 5 µl of 10 mM DHF to the cuvette already
containing 1x PBS, your GST-DHFR-His
sample and NADPH. Quickly cover the cuvette
with parafilm and invert 5 times.
G.
Immediately place the cuvette in the
spectrophotometer and begin kinetics run. As
run is proceeding, record absorbance value
every 15 seconds for 150 seconds. Remove
cuvette from the spectrophotometer.
+10 mM
DHF
Instrumentation
BioLogic LP
Demo
BioLogic™ LP
BioLogic DuoFlow™
54
Chromatography
instrumentation
1. Pump(s)
2. Detector(s)
-
UV detector
-
Conductivity detector
-
Pressure detector
-
Fluorescence detector
3. Valves
Plus associated wiring and tubing…
55
Examining a chromatogram
BioLogic LP
56
Examining a chromatogram
BioLogic DuoFlow
57
DHFR Enzymatic Activity Calculation
ΔOD, control
Slope of Control Data x 60 = _____________ Change in Absorbance at 340 nm/minute
ΔOD, reaction
Slope of Enzyme reaction data x 60 = _____________ Change in Absorbance at 340 nm/minute
ΔOD = |ΔOD, reaction| - |ΔOD, control|
ΔC (mol/liter/min) = ΔOD
εxl
58
ε (extinction coefficient) = 6220 M-1 cm-1 for NADPH
l (length) is the pathlength of the cuvette (usually 1 cm for most cuvettes)
Biomanufacturing
59
Scaling up of the process
developed during research and
development
Resources
and
References
 Bio-Rad:
Curriculum Training Specialists
[email protected]
http://explorer.bio-rad.com
Technical Support:
1(800)4BIORAD
[email protected]
 Northeast Biomanufacturing Center and
Collaborative (NBC2)
http://www.biomanufacturing.org
 Bio-Link (Elaine Johnson, Director)
http://www.bio-link.org
60
Jim DeKloe:
[email protected]
Protein
Expression
and
Purification
Series
Ordering
info
•166-5040EDU, Centrifugation
Process Series
•166-5045EDU, Handpacked
Column Process Series
(instrumentation)
•166-5050EDU, Prepacked
Cartridge Process Series
(instrumentation)
61
AVAILABLE NOW!
DHFR
Enzymatic
Assay
Module
SDS-PAGE
Electrophoresis
Module
Growth and
Expression
Module
Purification
Module
Option 3
Prepacked
Cartridge
Purification
Module
Option 1
Centrifugation
Purification
Module
Option 2
Handpacked
Column
Purification
Module