Protein Purification: From industrial enzymes to cancer therapy

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(No Transcript)
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Protein Purification From industrial enzymes to
cancer therapy
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Protein Expression and Purification
SeriesInstructors
Jim DeKloe Solano Community College james.dekloe_at_s
olano.edu Bio-Rad Curriculum and Training
Specialists Sherri Andrews, Ph.D. (Eastern
US) sherri_andrews_at_bio-rad.com Leigh Brown,
M.A. (Central US) leigh_brown_at_bio-rad.com Damon
Tighe (Western US) damon_tighe_at_bio-rad.com
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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

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Protein Expression and Purification Series
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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
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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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 Diabetes E. coli
Human growth hormone Growth disorders E. coli
Granulocyte colony stimulating factor Cancers E. Coli
Erythropoietin Anemia CHO cells
Tissue plasminogen activator Heart attack CHO cells
Hepatitis B virus vaccine Vaccination Yeast
Human papillomavirus vaccine Vaccination Yeast
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Biomanufacturing Defined
The production of pharmaceutical proteins using
genetically engineered cells
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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 Difficult Easy Easy
Deposition of product Intracellular Intracellular or extracellular Extracellular
Product Intracellular Often secreted into media Secreted
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DHFR Dihydrofolatereductase

• 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 chemotherapeutic agent
• -Competitive inhibitor of DHFR
• -Methotrexate resistance - correlates with
  amplification of DHFR genes

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Induction
Biotech companies genetically engineer plasmids
to place genes behind inducible promoters
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Transcriptional Regulation in the pDHFR system
Lactose
IPTG
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Transcriptional Regulation in the pDHFR system
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Transcriptional Regulation in the pDHFR system
Lactose Induced System
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GST-DHFR-His 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
• Target for chemotherapy reagents

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Selection Mechanism for Mammalian cells
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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 ? tail of Antibodies
• His-tagged ?metal complexes (Ni)
• Glutathione-s-transferase ? glutathione

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Performing the chromatographic separation

• Gravity Chromatography
• Spin Column Chromatography
• Chromatography Instrumentation
• Small scale
• Biomanufacturing scale
• (bioreactors)

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Protein Expression and Purification Series
Workflow
Streak Cells
Overnight culture
Subculture, monitor, and induce
Harvest and lyse cells
Analyze
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CentrifugeRCF 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
• Determine RPM for 1,000 x g. The Bio-Rad 16K
  microcentrifuge rotor has a radius of 7.3 cm

RCF relative centrifugal force RPM rotations
per minute R radius in cm from center of
rotor to middle of spin column
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Affinity purification

1. Label column with initials. Snap off bottom tab
   of column, remove cap and place in 2 ml
   microcentrifuge tube.

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

200 µl

1. Add 200 µl of Ni-IMAC resin slurry to empty column

Ni-IMAC resin slurry

1. Centrifuge for 2 minutes at 1,000 x g. After
   spin, discard buffer that has collected in the
   microcentrifuge tube.

discard
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Affinity purification
200 µl

1. Add 200 µl of distilled H2O to column

Distilled H2O

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

1. Centrifuge for 2 minutes at 1,000 x g. After
   spin, discard water from collection tube.

discard
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Affinity purification
500 µl

1. Add 500 µl of Equilibration buffer to column

Equilibration buffer

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

1. Centrifuge for 2 minutes at 1,000 x g. After
   spin, discard Equilibration buffer and collection
   tube. The column is now ready to use.

discard
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Affinity purification
600 µl

• Place yellow tip closure on bottom of column. Add
  600 µl Soluble Fraction to Column Put on clear
  top cap.

Soluble fraction

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

1. Gently mix for 20 min.

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His tags

• His tags are typically a series of 6 histidines
  added to the C or N terminus of a recombinant
  protein

Histidine

• His tag and column interaction

Resin
Ni
His-tagged Recombinant Protein
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His tags

• His and imidazole structure similarities
• Imidazole competes with His for Ni2 sites

Imidazole
Histidine
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Affinity purification

• Label three 2 ml tubes

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

Flow through Wash
Eluate
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Affinity purification

1. Remove yellow tip closure.
2. Place column in 2 ml collection tube labeled
   Flow Through and remove clear top cap.
3. Centrifuge for 2 min at 1,000x g. Set
   aside Flow Through.

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

Flow through
Keep
Flow through
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Affinity purification

• Place column in 2 ml collection tube labeled
  Wash.
• Add 600 µl Wash Buffer to column.
• Centrifuge for 2 min at 1,000xg. Set aside Wash
  fraction.

Wash

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

600 µl
Wash Buffer
Keep
Wash
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Affinity purification

• Place column in 2 ml collection tube labeled
  Eluate.
• Add 400 µl Elution Buffer to column.
• Centrifuge for 2 min at 1,000xg. Set aside Eluate.

Eluate

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

400 µl
Elution Buffer
Keep
Eluate
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Recap so far.
Soluble fraction
Started with a complex mixture of all the soluble
E. coli proteins along with the induced expressed
human GST-DHFR-His
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
Flowthrough Wash Eluate
600 µl 600 µl
400 µl
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Size exclusion purification(buffer exchange)
GST-DHFR-His in 20 mM sodium phosphate, 300 mM
NaCl and 250 mM imidazole
Eluate fraction
Imidazole
250 mM imidazole solution has an A280 0.2-0.4
W and Y contribute to A280 of proteins
NEED TO REMOVE IMIDAZOLE TO QUANTIFY PROTEIN
CONCENTRATION USING A280
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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

http//tainano.com/Molecular20Biology20Glossary.
files/image047.gif
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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

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SizeExclusion
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Size exclusion purification(desalting)

1. Label desalting column with your initials.
2. Invert column several times to resuspend gel.
3. Snap off bottom tip and place in a 2 ml
   collection tube.

1. Prepare SEC column
2. Desalt GST-DHFR-HIS with SEC column

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Size exclusion purification(desalting)

• 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.
• After draining, place column in clean 2 ml tube.
  
• Centrifuge for 2 min at 1,000 x g. Discard 2ml
  tube containing packing buffer.

discard

1. Prepare SEC column
2. Desalt GST-DHFR-HIS with SEC column

discard
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Size exclusion purification(desalting)
Removing the 250 mM imidazole solution by size
exclusion chromatography

1. Label new 2 ml tube Desalted Eluate.
2. Carefully apply 75 ul of eluate fraction directly
   to the center of column. Be careful not to touch
   resin with pipet tip.
3. Centrifuge for 4 min at 1,000 x g.
4. Repeat addition of 75 µl of Eluate fraction to
   column and centrifugation.

75 µl

1. Prepare SEC column
2. Desalt GST-DHFR-HIS with SEC column

Eluate
2 x
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Size exclusion purification(desalting)
Desalted eluate 150 µl GST-DHFR-His in 10 mM
Tris buffer 250 mM Imidazole has been removed

1. Prepare SEC column
2. Desalt GST-DHFR-HIS with SEC column

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Protein Analysis

• Determination of success of induction, lysis, and
  purification of GST-DHFR-His using SDS-PAGE
  analysis
• Measurement of concentration using the absorbance
  at 280 nm
• Enzymatic activity analysis

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Protein analysisSDS-PAGE
1 Precision Plus Dual Color
9 Desalted GST-DHFR-His
8 Eluted GST-DHFR-His
4 Insoluble fraction
2 Uninduced cells
5 Soluble fraction
6 Column flow through
standards
3 Induced cells
7 Column wash

1. Prepare Samples
2. Prepare TGX Gel and vertical Electrophoresis
   apparatus
3. Load and Run Gel
4. Stain gel
5. Analyze gel

250 150 100 75 50 37 25
20 15 10
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Protein analysis (Quantitation using A280)
Quantitation of Protein in Desalted Fraction
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.
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Protein analysis (Quantitation using A280)
Calculate concentration of GST-DHFR-His
Beers Law Aecl

• e - the molar absorptivity ((mol/L)-1 cm-1)
• l - the path length of the sample (usually
  1cm-cuvette)
• C - the concentration of the compound in
  solution (mol/L)

• For GST-DHFR-His
• 75,540 (mol/L)-1 cm-1
• C (mol/L) Absorbance
• 75,540 (mol/L)-1 cm-1 x 1 cm

Expected results 1.3 x 10-6 5.3 x 10-6 M
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Enzyme Assay
Absorbance at 340nm
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Enzyme Assay

1. Set up spectrophotometer for kinetics
   measurements at 340 nm.
2. Blanking the instrument. Add 985 µl 1x PBS to
   cuvette place in instrument, read as blank. Save
   cuvette with PBS
3. 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 GST-DHFR-His
   eluate to cuvette. Cover cuvette with parafilm
   and invert 10 times. Immediately place cuvette in
   spectrophotometer and begin kinetics run.
4. As run is proceeding, record absorbance value
   every 15 seconds for 150 seconds. Remove and save
   cuvette from the spectrophotometer.

985 µl 1x PBS
985 µl 1x PBS
15ul desalted Eluate
6 µl NADPH
985 µl 1x PBS
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Enzyme Assay

1. 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.
2. 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.
3. 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
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InstrumentationBioLogic LPDemo
BioLogic LP
BioLogic DuoFlow
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Chromatography instrumentation

• Pump(s)
• Detector(s)
• UV detector
• Conductivity detector
• Pressure detector
• Fluorescence detector
• Valves
• Plus associated wiring and tubing

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Examining a chromatogramBioLogic LP
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Examining a chromatogramBioLogic DuoFlow
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DHFR Enzymatic Activity Calculation
?OD, control
Slope of Control Data x 60 _____________ Change
in Absorbance at 340 nm/minute
?OD, reaction
Slope of Enzyme reaction data x 60
_____________ Change in Absorbance at 340
nm/minute
?OD ?OD, reaction - ?OD, control
?C (mol/liter/min) ?OD
e x l
e (extinction coefficient) 6220 M-1 cm-1 for
NADPH l (length) is the pathlength of the cuvette
(usually 1 cm for most cuvettes)
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Biomanufacturing
Scaling up of the process developed during
research and development
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• Bio-Rad
• Curriculum Training Specialists
• biotechnology_explorer_at_bio-rad.com
• http//explorer.bio-rad.com
• Technical Support
• 1(800)4BIORAD
• LSG_TechServ_US_at_bio-rad.com
• Northeast Biomanufacturing Center and
  Collaborative (NBC2)
• http//www.biomanufacturing.org
• Bio-Link (Elaine Johnson, Director)
• http//www.bio-link.org
• Jim DeKloe
• James.DeKloe_at_solano.edu

Resources and References
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ProteinExpressionandPurificationSeriesOrderin
g info

• AVAILABLE NOW!

• 166-5040EDU, Centrifugation Process Series
• 166-5045EDU, Handpacked Column Process Series
  (instrumentation)
• 166-5050EDU, Prepacked Cartridge Process Series
  (instrumentation)