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Protein Purification: From industrial enzymes to cancer therapy
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Protein Expression and Purification Series Instructors
Jim DeKloe Solano Community College Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. (Eastern US) Leigh Brown, M.A. (Central US) Damon Tighe (Western US)
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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
Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Option 2 Handpacked Column Growth and Expression SDS-PAGE Electrophoresis DHFR Enzymatic Assay
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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 Protein Expression and Purification Series Workshop Timeline
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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 High Cost of growth medium Product titer (concentration) Folding Sometimes Probably Yes Glycosylation No Yes, but different pattern Full Relative ease to grow Easy Difficult Relative ease of recovery Deposition of product Intracellular Intracellular or extracellular Extracellular Product Often secreted into media Secreted
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Protein – The product of Biotech
USED IN THE TREATMENT OF: Cell Production Insulin Diabetes E. coli Human growth hormone Growth disorders Granulocyte colony stimulating factor Cancers E. Coli Erythropoietin Anemia CHO cells Tissue plasminogen activator Heart attack Hepatitis B virus vaccine Vaccination Yeast Human papillomavirus vaccine
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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-DHFR-His Construct
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
RNA Polymerase Z Y A LacI Effector (Lactose) lac Operon 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 Protein Expression and Purification Series Workflow 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 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 1,000 3,497 7.3
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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 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 Distilled H2O 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 N3H+ -OOC Ni Resin Ni N NH Ni Ni N NH His-tagged Recombinant Protein
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His tags His and imidazole structure similarities
Imidazole competes with His for Ni2+ sites Histidine Imidazole N3H+ -OOC
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Affinity purification
Performing affinity chromatography Affinity purification 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 fraction Wash Buffer
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Affinity purification
Performing affinity chromatography (continued) Affinity purification 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= W and Y contribute to A280 of proteins NEED TO REMOVE IMIDAZOLE TO QUANTIFY PROTEIN CONCENTRATION USING A280
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Size Exclusion
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Size exclusion purification (buffer exchange)
Preparing the size exclusion column for usage Size exclusion purification (buffer exchange) 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 Size exclusion purification (buffer exchange) 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)
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)
Beer’s Law A=ecl Protein analysis (Quantitation using A280) 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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Instrumentation BioLogic LP Demo
BioLogic DuoFlow™
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Scaling up of the process developed during research and development
Biomanufacturing
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Resources and References
Bio-Rad: Curriculum Training Specialists Technical Support: 1(800)4BIORAD Northeast Biomanufacturing Center and Collaborative (NBC2) Bio-Link (Elaine Johnson, Director) Jim DeKloe:
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Protein Expression and Purification Series Ordering info
AVAILABLE SUMMER 2011 Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Option 2 Handpacked Column Growth and Expression SDS-PAGE Electrophoresis DHFR Enzymatic Assay EDU, Centrifugation Process Series EDU, Handpacked Column Process Series (instrumentation) EDU, Prepacked Cartridge Process Series (instrumentation)
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