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Protein Purification: From industrial enzymes to
cancer therapy
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Protein
Expression
and
Purification
Series
Jim DeKloe
Solano Community College
james.dekloe@solano.edu
Instructors
Bio-Rad Curriculum and Training Specialists:
Sherri Andrews, Ph.D. (Eastern US)
sherri_andrews@bio-rad.com
Leigh Brown, M.A. (Central US)
leigh_brown@bio-rad.com
Damon Tighe (Western US)
damon_tighe@bio-rad.com
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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
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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
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
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Protein
Expression
and
Purification
Series
Workshop
Timeline
• Introduction
• Recombinant protein expression and purification
for biomanufacturing
• Dihydrofolate reductase
• Affinity purification
• Perform affinity chromatography
• Perform size exclusion (desalting)
chromatography
• Quantitate purified protein
• Demonstration of BioLogic LP chromatography
instrument
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The Value
of Proteins
Price Per Gram
Bovine Growth Hormone
$14
Gold
$48
Insulin
$60
Growth Hormone
$227,000
Granulocyte Colony
Stimulating Factor
$1,357,000
*Prices in 2011 US Dollars
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Biomanufacturing
Defined
The production of
pharmaceutical proteins using
genetically engineered cells
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Expression
Choices
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Cell type:
• E. coli
• Yeast
• Mammalian
–CHO
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
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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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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
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DHFR and
Cancer
•DHFR inhibition or reduction disrupts nucleic acid
synthesis affecting
-Cell growth
-Proliferation
•Methotrexate – one of the first chemotherapeutic
agents
-Inhibits DHFR
-Methotrexate resistance - correlates with
amplification of DHFR genes
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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
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•Target for chemotherapy reagents
Induction
Biotech companies genetically
engineer plasmids to place
genes behind inducible
promoters
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Transcriptional
Regulation in
the pDHFR
system
lac Operon
LacI
Z
Y A
Effector (Lactose)
LacI
Z
Y A
RNA Polymerase
Z
Lactose
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Y A
IPTG
2 phases of
growth
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Recovery
Separation of protein from
other molecules
Purification
Separation of the protein of
interest from other proteins
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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.
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Types of Column
Chromatography
•Ion Exchange (protein charge)
•Size Exclusion (separates on size)
•Hydrophobic Interaction (hydrophobicity)
•Affinity:
•Protein A
•His-tagged
•Glutathione-s-transferase
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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
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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
•Pour column
•Wash resin to remove
packing buffer
Pouring a 100 µl Ni-IMAC column
Label column with initials.
Prepare column. Snap off
bottom tab of empty column,
remove cap and place in 2 ml
collection tube.
200 µl
•Equilibrate resin
•Bind GST-DHFR-His
•Elute unbound proteins
•Wash protein bound
onto the resin
•Elute GST-DHFR-His
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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
collection tube.
Ni-IMAC
resin
slurry
Affinity
purification
•Pour column
•Wash resin to
remove packing
buffer
•Equilibrate resin
Washing and equilibrating the 100 µl Ni-IMAC column
200 µl
Add 200 µl of distilled
H2O to column
Centrifuge for 2 minutes at
1,000 x g. After spin, discard
water from collection tube.
•Bind GST-DHFR-His
•Elute unbound proteins
•Wash protein bound
onto the resin
•Elute GST-DHFR-His
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Distilled
H2O
500 µl
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
Affinity
purification
•Pour column
•Wash resin to remove
packing buffer
Binding the GST-DHFR-His to the Ni-IMAC resin
600 µl
Place yellow tip closure on
bottom of column. Add 600 µl
Soluble Fraction to Column;
Put on clear top cap.
•Equilibrate resin
•Bind GST-DHFR-His
•Elute unbound proteins
•Wash protein bound
onto the resin
•Elute GST-DHFR-His
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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
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N3H+
Imidazole
Affinity
purification
•Pour column
•Wash resin to remove
packing buffer
•Equilibrate resin
•Bind GST-DHFR-His
•Elute unbound
proteins
•Wash protein bound
onto the resin
Performing affinity chromatography
Label three 2 ml tubes:
“Flow through”, “Wash” and “Eluate”.
Remove yellow tip closure. Place
column in 2 ml collection tube
labeled “Flow Through” and remove
clear top cap. Centrifuge for 2 min at
1,000 x g.
Set aside Flow Through.
Place column in 2 ml collection tube
labeled “Wash”. Add 600 µl Wash
Buffer to column.
•Elute GST-DHFR-His
Centrifuge for 2 min at 1,000 x g.
Set aside Wash fraction.
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Flow
through
fraction
600 µl
Wash
fraction
Wash
Buffer
Affinity
purification
•Pour column
•Wash resin to remove
packing buffer
Performing affinity chromatography (continued)
400 µl
Place column in 2 ml collection
tube labeled “Eluate”. Add 400
µl Elution Buffer to column.
Eluate
•Equilibrate resin
•Bind GST-DHFR-His
Centrifuge for 2 min at 1,000 x g.
•Elute unbound proteins
Set aside Eluate.
•Wash protein bound onto
the resin
Collected fractions
•Elute GST-DHFR-His
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Flow through
~600 µl
Wash
Eluate
~600 µl
~400 µl
Elution
Buffer
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
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W and Y contribute to
A280 of proteins
NEED TO REMOVE IMIDAZOLE TO QUANTIFY
PROTEIN CONCENTRATION USING A280
Size
Exclusion
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Size
exclusion
purification
(buffer
exchange)
Preparing the size exclusion column for usage
Label desalting column with your
initials. Prepare desalting column by
inverting sharply several times to
resuspend gel
Snap off tip and place in 2 ml
collection tube. Remove green top
cap.
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. After
draining, place column in clean 2 ml
tube.
Centrifuge for 2 min at 1,000 x g.
Discard remaining packing buffer and
collection tube.
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Size
exclusion
purification
(buffer
exchange)
Removing the 250 mM imidazole solution by size
exclusion chromatography
75 µl
Label new 2 ml tube Desalted
eluate. Carefully apply 75 ul of
Desalted
eluate fraction directly to the
center of column. Be careful not to eluate
touch resin with pipet tip.
Centrifuge for 4 min at 1,000 x g.
Collected fraction
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Desalted Eluate
~75 µl
Eluate
Protein
analysis
75 µl
Desalted
eluate
(Quantitation
using A280)
Set absorbance to 280 nm
Blank spec with distilled H2O
Measure absorbance of sample at 280nm
Print out your data
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Clean UV
cuvette
Beer’s Law
Protein
analysis
(Quantitation
using A280)
A=ecl
e - the molar absorbtivity
((mol/L)-1 cm-1)
l - the path length of the sample (usually 1cmcuvette)
C - the concentration of the compound in
solution (mol/L)
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
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Enzyme
Assay
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Instrumentation
BioLogic LP
Demo
BioLogic™ LP
BioLogic DuoFlow™
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Biomanufacturing
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Scaling up of the process
developed during research and
development
Resources
and
References
 Bio-Rad:
Curriculum Training Specialists
biotechnology_explorer@bio-rad.com
http://explorer.bio-rad.com
Technical Support:
1(800)4BIORAD
LSG_TechServ_US@bio-rad.com
 Northeast Biomanufacturing Center and
Collaborative (NBC2)
http://www.biomanufacturing.org
 Bio-Link (Elaine Johnson, Director)
http://www.bio-link.org
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Jim DeKloe:
James.DeKloe@solano.edu
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)
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AVAILABLE SUMMER 2011
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