2 Protein Purification: From industrial enzymes to cancer therapy 22
3 Protein Expression and Purification Series Instructors Jim DeKloeSolano Community CollegeBio-Rad Curriculum and Training Specialists:Sherri Andrews, Ph.D. (Eastern US)Leigh Brown, M.A. (Central US)Damon Tighe (Western US)
4 Why Teach about Protein Expression and Purification? Powerful teaching toolReal-world connectionsLink to careers and industryTangible resultsLaboratory extensionsInterdisciplinary – connects biochemistry, biomanufacturing, chemistry, biology and medical scienceMimics a complete workflow utilized in research and industry
5 Protein Expression and Purification Series Option 1CentrifugationPurificationModuleOption 3PrepackedCartridgeOption 2HandpackedColumnGrowth andExpressionSDS-PAGEElectrophoresisDHFREnzymaticAssay
6 Protein Expression and Purification Series Advantages Follows a complete workflow including bacterial cell culture, induction, fractionation, purification, and analysis of purified proteinTeaches affinity purificationWork with a non-colored protein that is comparable to real world applicationsIncludes ability to run at small scale using a 16k microcentrifuge or scaling up and using chromatography instrumentationPossibility of extensions including western blots, ELISAs, site-directed mutagenesis studies, induction experiments
7 Protein Expression and Purification Series Workshop Timeline IntroductionRecombinant protein expression and purification for biomanufacturingDihydrofolate reductaseAffinity purificationPerform affinity chromatographyPerform size exclusion (desalting) chromatographyQuantitate purified proteinDemonstration of BioLogic LP chromatography instrumentProtein Expression and Purification Series Workshop Timeline
8 The Value of Proteins Price Per Gram Bovine Growth Hormone $14 Gold $48Insulin$60Growth Hormone$227,000Granulocyte Colony Stimulating Factor$1,357,000*Prices in 2011 US Dollars
9 Biomanufacturing Defined The production of pharmaceutical proteins using genetically engineered cells
11 Expression Choices Parameter Bacteria Yeast Mammalian Contamination riskLowHighCost of growth mediumProduct titer (concentration)FoldingSometimesProbablyYesGlycosylationNoYes, but different patternFullRelative ease to growEasyDifficultRelative ease of recoveryDeposition of productIntracellularIntracellular or extracellularExtracellularProductOften secreted into mediaSecreted
12 Protein – The product of Biotech USED IN THE TREATMENT OF:Cell ProductionInsulinDiabetesE. coliHuman growth hormoneGrowth disordersGranulocyte colony stimulating factorCancersE. ColiErythropoietinAnemiaCHO cellsTissue plasminogen activatorHeart attackHepatitis B virus vaccineVaccinationYeastHuman papillomavirus vaccine
13 DHFR — Dihydrofolate reductase Converts dihydrofolate into tetrahydrofolate (THF) by the addition of a hydride from NADPHTHF is a methyl (CH3) group shuttle required for synthesis of essential molecules- nucleotides- amino acids
14 DHFR and CancerDHFR inhibition or reduction disrupts nucleic acid synthesis affecting-Cell growth-ProliferationMethotrexate – one of the first chemotherapeutic agents-Inhibits DHFR-Methotrexate resistance - correlates with amplification of DHFR genes
15 GST-DHFR-His Construct Glutathione-s-transferaseAdded to increase solubilityCan be used as a secondary purification methodologyHistidine tag6 Histidine tag that binds to certain metals such as nickelHuman dihydrofolate reductaseGene product of interestTarget for chemotherapy reagents
16 InductionBiotech companies genetically engineer plasmids to place genes behind inducible promoters
17 Transcriptional Regulation in the pDHFR system RNA PolymeraseZYALacIEffector (Lactose)lac OperonTranscriptional Regulation in the pDHFR systemLactoseIPTG
19 Recovery Separation of protein from other molecules Purification Separation of the protein of interest from other proteins
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 Types of Column Chromatography Ion Exchange (protein charge)Size Exclusion (separates on size)Hydrophobic Interaction (hydrophobicity)Affinity:Protein AHis-taggedGlutathione-s-transferase
23 Protein Expression and Purification Series Workflow Streak CellsProtein Expression and Purification Series WorkflowOvernight cultureSubculture, monitor, and induceHarvest and lyse cellsPurifyCentrifugation or InstrumentationAnalyze
24 Centrifuge RCF to RPM conversion Accurate RCF(g) is important for chromatography resinsRPM to RCF varies for different models of centrifuges due to variation in rotor radiusDetermine RPM for 1,000 x g. The Bio-Rad 16K microcentrifuge rotor has a radius of 7.3 cmRCF = relative centrifugal forceRPM = rotations per minuteR = radius in cm from center ofrotor to middle of spin column1,0003,4977.3
25 Affinity purification Pouring a 100 µl Ni-IMAC columnLabel column with initials. Prepare column. Snap off bottom tab of empty column, remove cap and place in 2 ml collection tube.Pour columnWash resin to remove packing bufferEquilibrate resinBind GST-DHFR-HisElute unbound proteinsWash protein bound onto the resinElute GST-DHFR-His200 µlNi-IMAC resin slurryAdd 200 µl of Ni-IMAC resin slurry to empty columnCentrifuge for 2 minutes at 1,000 x g. After spin, discard buffer that has collected in the collection tube.
26 Affinity purification Washing and equilibrating the 100 µl Ni-IMAC column200 µlAdd 200 µl of distilled H2O to columnPour columnWash resin to remove packing bufferEquilibrate resinBind GST-DHFR-HisElute unbound proteinsWash protein bound onto the resinElute GST-DHFR-HisDistilled H2OCentrifuge for 2 minutes at 1,000 x g. After spin, discard water from collection tube.500 µlAdd 500 µl of Equilibration buffer to columnEquilibrationbufferCentrifuge for 2 minutes at 1,000 x g. After spin, discard Equilibration buffer and collection tube. The column is now ready to use.
27 Affinity purification Binding the GST-DHFR-His to the Ni-IMAC resin600 µlPlace yellow tip closure on bottom of column. Add 600 µl Soluble Fraction to Column; Put on clear top cap.Pour columnWash resin to remove packing bufferEquilibrate resinBind GST-DHFR-HisElute unbound proteinsWash protein bound onto the resinElute GST-DHFR-HisSoluble fractionGently mix for 20 min.
28 His tagsHis tags are typically a series of 6 histidines added to the C or N terminus of a recombinant proteinHistidineHis tag and column interactionN3H+-OOCNiResinNiNNHNiNiNNHHis-taggedRecombinantProtein
29 His tags His and imidazole structure similarities Imidazole competes with His for Ni2+ sitesHistidineImidazoleN3H+-OOC
30 Affinity purification Performing affinity chromatographyAffinity purificationLabel 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.FlowthroughfractionPour columnWash resin to remove packing bufferEquilibrate resinBind GST-DHFR-HisElute unbound proteinsWash protein bound onto the resinElute GST-DHFR-His600 µlPlace 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.WashfractionWash Buffer
31 Affinity purification Performing affinity chromatography (continued)Affinity purification400 µlPlace column in 2 ml collection tube labeled “Eluate”. Add 400 µl Elution Buffer to column.Pour columnWash resin to remove packing bufferEquilibrate resinBind GST-DHFR-HisElute unbound proteinsWash protein bound onto the resinElute GST-DHFR-HisElution BufferEluateCentrifuge for 2 min at 1,000 x g.Set aside Eluate.Collected fractionsFlow through Wash Eluate~600 µl ~600 µl ~400 µl
32 Size exclusion purification (buffer exchange) GST-DHFR-His in 20 mM sodium phosphate, 300 mM NaCl and 250 mM imidazoleEluate fractionImidazole250 mM imidazole solution has an A280=W and Y contribute to A280 of proteinsNEED TO REMOVE IMIDAZOLE TO QUANTIFY PROTEIN CONCENTRATION USING A280
34 Size exclusion purification (buffer exchange) Preparing the size exclusion column for usageSize exclusion purification (buffer exchange)Label desalting column with your initials. Prepare desalting column by inverting sharply several times to resuspend gelSnap 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.
35 Size exclusion purification (buffer exchange) Removing the 250 mM imidazole solution by size exclusion chromatographySize exclusion purification (buffer exchange)75 µlLabel 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.DesaltedeluateEluateCollected fractionDesalted Eluate~75 µl
36 Protein analysis (Quantitation using A280) Clean UV cuvetteDesalted eluateSet absorbance to 280 nmBlank spec with distilled H2OMeasure absorbance of sample at 280nmPrint out your data
37 Protein analysis (Quantitation using A280) Beer’s LawA=eclProtein 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-1C (mol/L) = Absorbance75,540 (mol/L)-1 cm-1 x 1 cm
40 Scaling up of the process developed during research and development Biomanufacturing
41 Resources and References Bio-Rad:Curriculum Training SpecialistsTechnical Support:1(800)4BIORADNortheast Biomanufacturing Center and Collaborative (NBC2)Bio-Link (Elaine Johnson, Director)Jim DeKloe:
42 Protein Expression and Purification Series Ordering info AVAILABLE SUMMER 2011Option 1CentrifugationPurificationModuleOption 3PrepackedCartridgeOption 2HandpackedColumnGrowth andExpressionSDS-PAGEElectrophoresisDHFREnzymaticAssayEDU, Centrifugation Process SeriesEDU, Handpacked Column Process Series (instrumentation)EDU, Prepacked Cartridge Process Series (instrumentation)