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Protein Purification: From industrial enzymes to cancer therapy

Protein Purification: From industrial enzymes to cancer therapy. 2. Jim DeKloe Solano Community College james.dekloe@solano.edu Bio-Rad Curriculum and Training Specialists : Sherri Andrews, Ph.D. (Eastern US) sherri_andrews@bio-rad.com Leigh Brown, M.A. (Central US)

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Protein Purification: From industrial enzymes to cancer therapy

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  1. Protein Purification: From industrial enzymes to cancer therapy 2

  2. Jim DeKloe Solano Community College james.dekloe@solano.edu 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 Protein Expression and Purification SeriesInstructors

  3. 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

  4. 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

  5. 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

  6. 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

  7. The Value of Proteins Price Per Gram Prices in 2011 US Dollars * As of 8/14/2011

  8. Protein – The product of Biotech

  9. Biomanufacturing Defined The production of pharmaceutical proteins using genetically engineered cells

  10. Expression Choices Cell type: • E. coli • Yeast • Mammalian • CHO

  11. Expression Choices

  12. 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

  13. 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

  14. Induction Biotech companies genetically engineer plasmids to place genes behind inducible promoters

  15. 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

  16. Transcriptional Regulation in the pDHFR system

  17. Transcriptional Regulation in the pDHFR system Lactose = Induced System

  18. 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

  19. Selection Mechanism for Mammalian cells

  20. Phases of growth

  21. Recovery Separation of protein from other molecules Purification Separation of the protein of interest from other proteins

  22. 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.

  23. 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

  24. Performing the chromatographic separation • Gravity Chromatography • Spin Column Chromatography • Chromatography Instrumentation • Small scale • Biomanufacturing scale • (bioreactors)

  25. Purify Centrifugation or Instrumentation Streak Cells Protein Expression and Purification Series Workflow Overnight culture Subculture, monitor, and induce Harvest and lyse cells Analyze

  26. 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

  27. Affinity purification • Label column with initials. Snap off bottom tab of column, remove cap and place in 2 ml microcentrifuge 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 • Add 200 µl of Ni-IMAC resin slurry to empty column Ni-IMAC resin slurry • Centrifuge for 2 minutes at 1,000 x g. After spin, discard buffer that has collected in the microcentrifuge tube. discard

  28. Affinity purification 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. discard

  29. Affinity purification 500 µl • Add 500 µl of Equilibration buffer to column Equilibration buffer • 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 Equilibration buffer and collection tube. The column is now ready to use. discard

  30. 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 • 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 • Gently mix for 20 min.

  31. 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

  32. -OOC N3H+ • His and imidazole structure similarities • Imidazole competes with His for Ni2+ sites His tags Histidine Imidazole

  33. Affinity purification • Label three 2 ml tubes: • 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 “Flow through” “Wash” “ Eluate”

  34. Affinity purification • Remove yellow tip closure. • Place column in 2 ml collection tube labeled “Flow Through” and remove clear top cap. • Centrifuge for 2 min at 1,000x g. Set aside Flow Through. • 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 “Flow through” Keep “Flow through”

  35. Affinity purification • Place column in 2 ml collection tube labeled “Wash”. • Add 600 µl Wash Buffer to column. • Centrifuge for 2 minat 1,000xg. Set aside Wash fraction. “Wash” • 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 Wash Buffer Keep “Wash”

  36. Affinity purification • Place column in 2 ml collection tube labeled “Eluate”. • Add 400 µl Elution Buffer to column. • Centrifuge for 2 minat 1,000xg. Set aside Eluate. “Eluate” • 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 400 µl Elution Buffer Keep “Eluate”

  37. Soluble fraction Recap so far…. 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

  38. 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

  39. 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 • Large molecules move through the column quickly traveling around the beads • Smaller molecules move through the pores of the beads and take longer to pass through the column http://tainano.com/Molecular%20Biology%20Glossary.files/image047.gif

  40. 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

  41. SizeExclusion

  42. Size exclusion purification(desalting) • Label desalting column with your initials. • Invert column several times to resuspend gel. • Snap off bottom tip and place in a 2 ml collection tube. • Prepare SEC column • Desalt GST-DHFR-HIS with SEC column

  43. 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 • Prepare SEC column • Desalt GST-DHFR-HIS with SEC column discard

  44. Removing the 250 mM imidazole solution by size exclusion chromatography Size exclusion purification(desalting) • 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. • Repeat addition of 75 µl of Eluate fraction to column and centrifugation. 75 µl • Prepare SEC column • Desalt GST-DHFR-HIS with SEC column “Eluate” 2 x

  45. Size exclusion purification(desalting) Desalted eluate ~150 µl GST-DHFR-His in 10 mM Tris buffer 250 mM Imidazole has been removed • Prepare SEC column • Desalt GST-DHFR-HIS with SEC column

  46. 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

  47. 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 • Prepare Samples • Prepare TGX Gel and vertical Electrophoresis apparatus • Load and Run Gel • Stain gel • Analyze gel 250 150 100 75 50 37 25 20 15 10

  48. +100 µl Distilled H2O Clean UV cuvette + 100 µl Desalted eluate Clean UV cuvette Quantitation of Protein in Desalted Fraction Protein analysis(Quantitation using A280) 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.

  49. Calculate concentration of GST-DHFR-His Protein analysis(Quantitation using A280) Beer’s Law A=ecl 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-1x 1 cm Expected results 1.3 x 10-6 – 5.3 x 10-6 M

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