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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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
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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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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

• 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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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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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 one of the first chemotherapeutic
  agents
• -Inhibits DHFR
• -Methotrexate resistance - correlates with
  amplification of DHFR genes

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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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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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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

• 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
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.

• Pour column
• Wash resin to remove packing buffer
• Equilibrate resin
• Bind GST-DHFR-His
• Elute unbound proteins
• Wash protein bound onto the resin
• Elute GST-DHFR-His

200 µl
Ni-IMAC resin slurry
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.
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Affinity purification
Washing and equilibrating the 100 µl Ni-IMAC
column
200 µl
Add 200 µl of distilled H2O to column
Distilled H2O

• Pour column
• Wash resin to remove packing buffer
• Equilibrate resin
• Bind GST-DHFR-His
• Elute unbound proteins
• Wash protein bound onto the resin
• Elute GST-DHFR-His

Centrifuge for 2 minutes at 1,000 x g. After
spin, discard water from collection tube.
500 µl
Add 500 µl of Equilibration buffer to column
Equilibration buffer
Centrifuge for 2 minutes at 1,000 x g. After
spin, discard Equilibration buffer and collection
tube. The column is now ready to use.
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Affinity purification
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.

• Pour column
• Wash resin to remove packing buffer
• Equilibrate resin
• Bind GST-DHFR-His
• Elute unbound proteins
• Wash protein bound onto the resin
• Elute GST-DHFR-His

Soluble fraction
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
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.
Flow through fraction

• Pour column
• Wash resin to remove packing buffer
• Equilibrate resin
• Bind GST-DHFR-His
• Elute unbound proteins
• Wash protein bound onto the resin
• Elute GST-DHFR-His

600 µl
Place column in 2 ml collection tube labeled
Wash. Add 600 µl Wash Buffer to column.
Centrifuge for 2 min at 1,000 x g. Set aside
Wash fraction.
Wash Buffer
Wash fraction
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Affinity purification
Performing affinity chromatography (continued)
400 µl
Place column in 2 ml collection tube labeled
Eluate. Add 400 µl Elution Buffer to column.

• Pour column
• Wash resin to remove packing buffer
• Equilibrate resin
• Bind GST-DHFR-His
• Elute unbound proteins
• Wash protein bound onto the resin
• Elute GST-DHFR-His

Elution Buffer
Eluate
Centrifuge for 2 min at 1,000 x g. Set aside
Eluate.
Collected fractions
Flow through 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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SizeExclusion
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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 eluate fraction directly to the
center of column. Be careful not to touch resin
with pipet tip. Centrifuge for 4 min at 1,000 x
g.
Desalted eluate
Eluate
Collected fraction
Desalted Eluate 75 µl
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Protein analysis (Quantitation using A280)
75 µl
Clean UV cuvette
Desalted eluate
Set absorbance to 280 nm Blank spec with
distilled H2O Measure absorbance of sample at
280nm Print out your data
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Protein analysis (Quantitation using A280)
Beers Law Aecl

• e - the molar absorbtivity ((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

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Enzyme Assay
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InstrumentationBioLogic LPDemo
BioLogic LP
BioLogic DuoFlow
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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 SUMMER 2011

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