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

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Presentation on theme: "1. 2 2 Protein Purification: From industrial enzymes to cancer therapy."— Presentation transcript:

1 1

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

3 3 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)

4 4 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

5 5 Protein Expression and Purification Series Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Purification Module Option 2 Handpacked Column Purification Module Growth and Expression Module SDS-PAGE Electrophoresis Module DHFR Enzymatic Assay Module Purification Module

6 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 7 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

8 8 The Value of Proteins Bovine Growth Hormone$14 Gold$48 Insulin$60 Growth Hormone$227,000 Granulocyte Colony Stimulating Factor $1,357,000 Price Per Gram *Prices in 2011 US Dollars

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

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

11 11 Expression Choices ParameterBacteriaYeastMammalian Contamination riskLow High Cost of growth medium Low High Product titer (concentration) High Low FoldingSometimesProbablyYes GlycosylationNoYes, but different patternFull Relative ease to growEasy Difficult Relative ease of recovery DifficultEasy Deposition of productIntracellularIntracellular or extracellularExtracellular ProductIntracellularOften secreted into mediaSecreted

12 12 PROTEIN: USED IN THE TREATMENT OF: Cell Production InsulinDiabetesE. coli Human growth hormoneGrowth disordersE. coli Granulocyte colony stimulating factorCancersE. Coli ErythropoietinAnemiaCHO cells Tissue plasminogen activatorHeart attackCHO cells Hepatitis B virus vaccineVaccinationYeast Human papillomavirus vaccineVaccinationYeast Protein – The product of Biotech

13 13 DHFR Dihydrofolate reductase Converts dihydrofolate into tetrahydrofolate (THF) by the addition of a hydride from NADPH THF is a methyl (CH 3 ) group shuttle required for synthesis of essential molecules - nucleotides - amino acids

14 14 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

15 15 GST-DHFR- His Construct GST – DHFR - His Glutathione-s-transferase Added to increase solubility Can be used as a secondary purification methodology Human dihydrofolate reductase Gene product of interest Target for chemotherapy reagents Histidine tag 6 Histidine tag that binds to certain metals such as nickel

16 16 Induction Biotech companies genetically engineer plasmids to place genes behind inducible promoters

17 17 Transcriptional Regulation in the pDHFR system RNA Polymerase ZYA ZYA LacI Effector (Lactose) ZYA LacI lac Operon LactoseIPTG

18 18 2 phases of growth

19 19 Recovery Separation of protein from other molecules Purification Separation of the protein of interest from other proteins

20 20 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.

21 21 Types of Column Chromatography Ion Exchange (protein charge) Size Exclusion (separates on size) Hydrophobic Interaction (hydrophobicity) Affinity: Protein A His-tagged Glutathione-s-transferase

22 22 Performing the chromatographic separation Gravity Chromatography Spin Column Chromatography Chromatography Instrumentation Small scale Biomanufacturing scale (bioreactors)

23 23 Protein Expression and Purification Series Workflow Streak Cells Overnight culture Subculture, monitor, and induce Harvest and lyse cells Purify Centrifugation or Instrumentation Analyze

24 24 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 1,0003,

25 25 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. 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. Ni-IMAC resin slurry 200 µl 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

26 26 Affinity purification Add 200 µl of distilled H 2 O to column Centrifuge for 2 minutes at 1,000 x g. After spin, discard water from collection tube. Add 500 µl of Equilibration buffer to column Centrifuge for 2 minutes at 1,000 x g. After spin, discard Equilibration buffer and collection tube. The column is now ready to use. Washing and equilibrating the 100 µl Ni-IMAC column Distilled H 2 O 200 µl Equilibration buffer 500 µl 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

27 27 Affinity purification Gently mix for 20 min. Place yellow tip closure on bottom of column. Add 600 µl Soluble Fraction to Column; Put on clear top cap. Soluble fraction Binding the GST-DHFR-His to the Ni-IMAC resin 600 µl 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

28 28 His tags N3H+N3H+- OOC Histidine Resin His tags are typically a series of 6 histidines added to the C or N terminus of a recombinant protein Ni NNH N His-tagged Recombinant Protein His tag and column interaction

29 29 His tags Imidazole N3H+N3H+- OOC Histidine His and imidazole structure similarities Imidazole competes with His for Ni 2+ sites

30 30 Affinity purification Performing affinity chromatography 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. 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 Wash Buffer Wash fraction 600 µl 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

31 31 Affinity purification Performing affinity chromatography (continued) Elution Buffer Eluate Centrifuge for 2 min at 1,000 x g. Set aside Eluate. Place column in 2 ml collection tube labeled Eluate. Add 400 µl Elution Buffer to column. 400 µl Flow through Wash Eluate ~600 µl ~600 µl ~400 µl Collected fractions 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

32 32 Size exclusion purification (buffer exchange) Eluate fraction GST-DHFR-His in 20 mM sodium phosphate, 300 mM NaCl and 250 mM imidazole Imidazole 250 mM imidazole solution has an A 280 = W and Y contribute to A 280 of proteins NEED TO REMOVE IMIDAZOLE TO QUANTIFY PROTEIN CONCENTRATION USING A 280

33 33 Size Exclusion

34 34 Size exclusion purification (buffer exchange) Label desalting column with your initials. Prepare desalting column by inverting sharply several times to resuspend gel Centrifuge for 2 min at 1,000 x g. Discard remaining packing buffer and collection tube. 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. Preparing the size exclusion column for usage

35 35 Size exclusion purification (buffer exchange) Eluate Desalted eluate 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. 75 µl Removing the 250 mM imidazole solution by size exclusion chromatography Collected fraction Desalted Eluate ~75 µl

36 36 Protein analysis (Quantitation using A 280 ) Desalted eluate Clean UV cuvette Set absorbance to 280 nm Blank spec with distilled H 2 O Measure absorbance of sample at 280nm Print out your data 75 µl

37 37 Beers Law A= cl - 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 Protein analysis (Quantitation using A 280 )

38 38 Enzyme Assay

39 39 Instrumentation BioLogic LP Demo BioLogic LP BioLogic DuoFlow

40 40 Biomanufacturing Scaling up of the process developed during research and development

41 41 Bio-Rad: Curriculum Training Specialists Technical Support: 1(800)4BIORAD Northeast Biomanufacturing Center and Collaborative (NBC2) Bio-Link (Elaine Johnson, Director) Jim DeKloe: Resources and References

42 42 AVAILABLE SUMMER 2011 Protein Expression and Purification Series Ordering info Option 1 Centrifugation Purification Module Option 3 Prepacked Cartridge Purification Module Option 2 Handpacked Column Purification Module Growth and Expression Module SDS-PAGE Electrophoresis Module DHFR Enzymatic Assay Module Purification Module EDU, Centrifugation Process Series EDU, Handpacked Column Process Series (instrumentation) EDU, Prepacked Cartridge Process Series (instrumentation)


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