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Protein Purification: From industrial enzymes to cancer therapy . 2. Jim DeKloe Solano Community College firstname.lastname@example.org Bio-Rad Curriculum and Training Specialists : Sherri Andrews, Ph.D. (Eastern US) email@example.com Leigh Brown, M.A. (Central US)
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Jim DeKloe Solano Community College firstname.lastname@example.org Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. (Eastern US) email@example.com Leigh Brown, M.A. (Central US) firstname.lastname@example.org Damon Tighe (Western US) email@example.com Protein Expression and Purification SeriesInstructors
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
DHFR Enzymatic Assay Module SDS-PAGE Electrophoresis Module Growth and Expression Module Purification Module Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Purification Module Option 2 Handpacked Column Purification Module Protein Expression and Purification Series
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
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
The Value of Proteins Price Per Gram *Prices in 2011 US Dollars
Biomanufacturing Defined The production of pharmaceutical proteins using genetically engineered cells
Expression Choices Cell type: • E. coli • Yeast • Mammalian • CHO
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
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
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
Induction Biotech companies genetically engineer plasmids to place genes behind inducible promoters
lac Operon LacI Z Y A Effector(Lactose) LacI Z Y A RNA Polymerase Z Y A Transcriptional Regulation in the pDHFR system Lactose IPTG
Recovery Separation of protein from other molecules Purification Separation of the protein of interest from other proteins
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.
Types of Column Chromatography • Ion Exchange (protein charge) • Size Exclusion (separates on size) • Hydrophobic Interaction (hydrophobicity) • Affinity: • Protein A • His-tagged • Glutathione-s-transferase
Performing the chromatographic separation • Gravity Chromatography • Spin Column Chromatography • Chromatography Instrumentation • Small scale • Biomanufacturing scale • (bioreactors)
Purify Centrifugation or Instrumentation Streak Cells Protein Expression and Purification Series Workflow Overnight culture Subculture, monitor, and induce Harvest and lyse cells Analyze
3,497 1,000 7.3 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
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.
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.
Binding the GST-DHFR-His to the Ni-IMAC resin Affinity purification 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.
Ni Ni Ni -OOC N3H+ N N NH NH His tags • 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 Histidine Resin Ni His-tagged Recombinant Protein
-OOC N3H+ • His and imidazole structure similarities • Imidazole competes with His for Ni2+ sites His tags Histidine Imidazole
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 minat 1,000 x g. Set aside Wash fraction. Wash Buffer Wash fraction
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
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
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.
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
75 µl 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
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-1x 1 cm
InstrumentationBioLogic LPDemo BioLogic™ LP BioLogic DuoFlow™
Biomanufacturing Scaling up of the process developed during research and development
Resources and References • Bio-Rad: • Curriculum Training Specialists • firstname.lastname@example.org • 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 • Jim DeKloe: James.DeKloe@solano.edu
DHFR Enzymatic Assay Module SDS-PAGE Electrophoresis Module Growth and Expression Module Purification Module Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Purification Module Option 2 Handpacked Column Purification Module ProteinExpressionandPurificationSeriesOrdering info AVAILABLE SUMMER 2011 • 166-5040EDU, Centrifugation Process Series • 166-5045EDU, Handpacked Column Process Series (instrumentation) • 166-5050EDU, Prepacked Cartridge Process Series (instrumentation)