5_ProteinPurification(1)
Protein Expression and purification
Protein Expression and Purification22,159–164(2001)160SCOTT A.LESLEYTABLE1Genomic versus Proteomic TechnologiesGenomic technologies(DNA)Proteomic technologies(protein) Identification Determined experimentally,bioinformatics Predicted from genomic informationFunction1-dimensional information storage3-dimensional organization of chemicalfunctionalitiesBuilding blocks4bases20ϩamino acidsDetection sensitivity PCR amplification techniques Direct detection methodsSynthetic approaches Cheap and efficient oligonucleotide synthesis Limited capacity of peptide synthesismethods combined with PCRSequence determination500–700bases common by automated sequencing Direct sequencing difficult,mass spectrometry Purification Generic methods Generic methods require modification of proteinthrough gene fusionAnalysis methods Typically employ enzymes,hybridization Chemical,biophysical,biochemicaland mutagenesis),interactions between proteins(two-are of primary interest and typically are expressed in hybrid),and global protein changes(2D gels and LC–a bacterial host.Often this approach leads to problems MS).Purified protein is often required in these studies associated with expression levels and proper folding of and defines the outputs of any parallel expression and the protein of interest.Flexibility in expression options purification process.is a key parameter.Pichia or baculovirus expressionsystems can offer effective alternatives to bacterial sys-tems.Each expression scenario requires a specific vec-GENE CLONING FOR EXPRESSIONtor.Recloning cDNAs into each of these specific vectors Determining gene function through genomics typi-is extremely labor-intensive.Recombinatorial cloning cally starts from a query of a database.Sequence infor-methods provide an opportunity to minimize the effort mation for the3.9billion bases of sequence from the required for alternate expression.human genome is now available(1,2).Access and inter-Two systems are commonly used for recombinatorial pretation of this information often require sophisticated cloning and shown in Fig.1.The cre–lox recombination bioinformatics software outside the scope of this dis-system described by Elledge utilizes a single recombina-cussion.Public archives such as the Unigene Data-tion to introduce the gene of interest into a recipient base(/)or TIGR(http://vector(3).This is an in vitro reaction combined with a /)provide bioinformatic access to many in-genetic selection for the recombinant vector.In this way teresting plete genomic sequence infor-the gene of interest is cloned once into a donor vector mation is now available through Celera(http://and can then be moved into any of a number of recipient /index.cfm).Future annotation of fullplasmids for expression in different hosts or to utilize sequence information will greatly expand the access todifferent purification tags.A similar system utilizes full-length cDNA sequences.The first requirement is converting this genomic in-lambda Int/Xis/IHF recombination at att sites(4)to formation into an actual cDNA clone of that gene.Am-achieve transfer of open reading frames(ORFs).This plification of full-length cDNAs via PCR is the typical system has the advantage of a precise ORF transfer to first step.Both reverse transcriptase and amplification the expression vector rather than the cointegrant vector polymerases typically are lacking in proofreading activ-product of the cre–lox system.With either system,the ity.Care must be taken to limit the number of steps of primary limitation is that translational fusion of the amplification and to use proofreading enzymes where recombination sites is typically required to maintain possible to minimize the probability of introducing un-the flexibility and utility of the recombination method wanted mutations.Tissue selection for cDNA librariesfor expression.In those cases,such as crystallography, also is an important consideration for attempting towhere translational fusions are potentially detrimental isolate genes as they must be expressed within thatto the protein,a conventional cloning approach is library source for successful amplification.This infor-more appropriate.mation is often obtained from cDNA or oligonucleotideRegardless of the cloning method,parallel expression expression arrays.and purification requires utilization of purification Amplified gene products are cloned into appropriatetags.Many options exist for this purpose.A comprehen-vectors for expression.Depending on the source of thesive review is beyond the scope of this article.A list of gene,the host,and the end use of the protein,manydifferent vectors may be appropriate.Eukaryotic genes some commonly used tags is shown in Table2.By farHIGH-THROUGHPUT PROTEOMICS161FIG.1.Strategies of recombinatorial cloning.Individual cDNAs are cloned into a donor vector that can then be recombined into any number of recipient vectors through recombinatorial cloning.One option is to form a cointegrant plasmid through Cre-mediated recombination across a lox site.In the second scenario,a cDNA is flanked by phage lambda att sites which direct recombination into an expression vector through the use of the INT/XIS/IHF proteins.In these ways,a single donor clone can easily be transferred into any number of recipient vectors. the most common fusion is the histidine tag for purifica-analysis studies into small-scale for analysis of expres-tion on metal-chelate resins.This tag provides a sub-sion levels and properties and into large-scale for use stantial purification handle while being relatively un-with many of the proteomic applications.obtrusive as a fusion partner.Beyond purification,Small-scale expression is most useful for identifying translational fusions often provide a means to enhance those clones which express recombinant protein to high expression.The larger fusion tags such as thioredoxin levels and for evaluating the folding state of the protein. and GST often are superior in this respect.Crude expression testing is typically done by simpleSDS–PAGE analysis of whole cells.Evaluation of thefolding state is typically done by centrifugal fraction-RECOMBINANT EXPRESSION OF PROTEINation of a lysate,requiring a more gentle lysis proce-dure.Several lysozyme and mild detergent methods are Bacterial expression is most common for recombinantcommercially available for this purpose.proteins because of its ease of use and the high levelsof protein obtained.It is useful to divide expression For large-scale production for many applications,TABLE2Common Purification TagsBasis of purification Elution Reference Small tagsHistidine tag Metal affinity resin Imidizole(5)S-tag Interaction with S-protein Temperature(6) Calmodulin-binding protein Interaction with Calmodulin Calcium(7) Large tagsGlutathione S-transferase Glutathione agarose Reduced glutathione(8) Thioredoxin Phenylarsine oxide resin-Mercaptoethanol(9) Biotinylation domain Monomeric avidin resin Biotin(10) Maltose binding protein Amylose resin Maltose(11) Chitin binding protein/intein Chitin affinity Thiol(12)162SCOTT A.LESLEYtens of milligrams of protein typically are required.PURIFICATION STRATEGYEven with common bacterial expression levels,500–Proteins are highly diverse in their properties mak-1000ml of culture typically is required to provide these ing generic methods of purification difficult.Purifica-amounts.While such methods are commonplace in labo-tion tags such as described in Table2are a typical ratories,systems for parallel processing large numbers solution for purifying proteins in parallel.His-tag fu-of cultures at this level are not commercially available.sions are very common and provide a single-step chro-By developing instrumentation and optimizing media matographic purification that yields protein of suffi-and aeration conditions for high-density cell growth,cient purity for most applications.In addition,the his-our laboratory can parallel process96cultures at this tag sequence requires the addition of only six amino scale.Optical density(OD)values at600nm can reach acids to the recombinant protein,reducing the likeli-a value of40with logarithmic growth through at least hood that such a fusion will adversely affect gene func-30OD units.These cell densities allow us to producetion.A typical purification strategy is outlined in Fig.2. sufficient cell mass with65-ml culture volumes to yieldtens of milligrams of recombinant protein,sufficient for PURIFICATION AUTOMATIONmost applications.Such instrumentation is not com-Parallel processing typically involves instrumenta-monly available,but common shaking incubators cantion for automation.Lysis methods such as sonication or substitute with larger volumes.using a French press are not simple automation tasks. Recombinant expression of proteins is achievedLikewise,centrifugation is not easily integrated into through induction of a strong promoter system.Manyautomation due to problems of locating and indexing options exist in this regard including tac,T7,lambdathe rotor position.Automation of this process typically P L,and ara B promoters.It is important for parallelinvolves protocol modifications.This can easily be processing that growth and induction characteristicsachieved in small-scale methods.are consistent.For this reason,it is important to retainOn small-scale,parallel processing usually involves tight repression of expression and have a simple induc-use of a96-well plate format.Lysis is typically achieved tion procedure for high-level expression.For T7sys-using a combination of lysozyme and freeze–thaw cy-tems,the lac operator and T7lysozyme(pLysS)provide cles.Phage lysozymes are more effective than hen egg an extra level of repression.The arabinose promoter is white lysozyme for this purpose and can be combined tightly repressed in the absence of inducer and is our with nucleases to reduce viscosity and facilitate re-preferred system for parallel growth.With all of the moval of cell debris at the low g forces commonly used promoters listed,recombinant expression levels of10–with microtiter plates.Alternatively,nonionic deter-50%of total cell protein are common.gents can be employed for nondenaturing lysis.FIG.2.Generalized purification strategy of recombinant fusion protein.A common purification strategy is shown here.Proteins are purified from fermentation cultures by affinity purification.Isolated cell pellets are resuspended in an appropriate lysis buffer and disrupted by high-intensity sonication.Cell walls and insoluble debris are pelleted by centrifugation and the soluble supernatant containing the recombinant fusion protein is applied to chromatography resin containing an immobilized metal for affinity purification.Fractions containing the recombinant protein can be used directly or further purified using conventional chromatographic techniques.HIGH-THROUGHPUT PROTEOMICS163TABLE3Robotic Systems with Capabilities Adaptable to ProteinPurificationManu-facturer Instrument WebsiteQiagen BioRobot3000/Tomtec Quadra96/Matrix PlateMate /Hamilton Microlab4200/Beckman Biomek2000/Packard PlateTrak /index2.htmGilson Nebula215/index.htmlRobotic systems for nucleic acid purification are rela-tively commonplace and have recently been adapted forprotein purification.The Qiagen BioRobot3000per-forms multiple functions relevant to protein purifica-tion.It provides aspirate,dispense,pipet,vacuum fil-tration,and plate-shaking functions on a relativelycompact platform.These functions can be adapted to FIG.4.SDS–PAGE analysis of purified protein.Metal affinity chro-perform cell lysis and chromatography steps from1–2matography yields highly purified protein from a single chromato-graphic separation.This gel shows typical yields and purity obtained ml of bacterial culture.Specialized96-well plates clearfrom parallel purification using an automated purification system. cell debris via vacuum filtration and are also used toSuch proteins have been incorporated directly in successful crystalli-retain resin for chromatographic separations.The Wal-zation trials.Ten-microliter samples of12ml protein eluates from Ni-lac Quadra96also has most of these capacities and resin were separated by10%SDS–PAGE.Samples are recombinant can parallel process96or384samples.Both of thesefusions of thioredoxin to human proteins as indicated by accessionnumber.systems have been used with success in our laboratoryfor small-scale protein purification of proteins in micro-titer plates.Table3lists some robotic systems that maybe applied to small-scale protein purification.providing the throughput needed for proteomic studies Despite the difficulties,large-scale protein purifica-involving tens of thousands of proteins.We are cur-tion also can be automated.In our laboratory we simul-rently able to process approximately96–192proteins taneously process96bacterial cultures of65–70ml.per day with this system with average yields of around Instrumentation for processing96parallel cultures at10mg of purified protein.Affinity purification results that scale required development of custom roboticsin recombinant protein that is typically80–90%pure shown in Fig.3.These robotics incorporate liquid aspi-(see Fig.4)which is sufficient for most applications. rate and dispense,centrifugation,and sonication capa-Subsequent purification is sometimes necessary,for ex-bilities required for purification.Automation is key to ample,in protein crystallography,and is achieved usingFIG.3.Protein purification automation.Custom robotics for performing the purification strategy outlined in Fig.2are shown.(a)The instrument has capacity for automated liquid aspiration and dispensing,sonication,centrifugation,and fractionation.Ninety-six cultures are processed in parallel,giving up to10–50mg of purified protein per culture.(b)Expanded view of aspirate/dispense/sonicate head accessing rotor.164SCOTT A.LESLEYREFERENCESstandard ion-exchange and size-exclusion chromatog-raphy.Automation of these methods is relatively1.Venter,J.C.et al.(2001)The sequence of the human genome. straightforward employing standard FPLC and au-Science291,1304–1351.tosampler instrumentation.2.International Human Genome Sequencing Consortium(2001)Initial sequencing and analysis of the human genome.Nature SUMMARY409,860–921.3.Liu,Q.,Li,M.Z.,Leibham,D.,Cortez,D.,and Elledge,S.J. Determining gene function and understanding the(1999)The univector plasmid-fusion system,a method for rapid relationships and interactions of the gene products are construction of recombinant DNA without restriction enzymes.a global effort in biological studies.The approach toCurr.Biol.8,1300–1309.performing this immense task is driven by the availabil- 4.Hatley,J.L.,Temple,G.F.,and Brasch,M.A.(2000)DNA cloningity of genomic information.To utilize this informationusing in vivo site-specific recombination.Genome Res.10,pp.1788–1795.for experimentation,however,significant effort isneeded to actually isolate and express proteins from5.Petty,K.J.(1996)Metal-chelate affinity chromatography,in“Current Protocols in Molecular Biology,”Vol.2,Wiley,New York. the genes of interest for study.The complexity of this6.Kim,J.S.,and Raines,R.T.(1993)Ribonuclease S-peptide as a effort is compounded by the large number of gene prod-carrier in fusion proteins.Protein Sci.2,348–356.ucts comprising the proteome.Parallel processing and7.Stofko-Hahn,R.E.,.Carr,D.W,and Scott,J.D.(1992)A single generic methods are required to achieve a systematicstep purification for recombinant proteins.FEBS Lett.302, and thorough evaluation of gene function.274–278.Experimental uses of proteins for structural and8.Smith,D.B.,and Johnson,K.S.(1988)Single-step purification functional studies typically require milligram amounts of polypeptides expressed in Escherichia coli as fusions within purified form.Unlike genomic technologies that pri-glutathione S-transferase.Gene67,31–40.marily involve the study of nucleic acids,proteomic9.Lu,Z.,DiBlasio-Smith,E.A.,Grant,K.L.,Warne,N.W.,LaVallie,studies focus on proteins.Proteins are by nature much E.R.,Collins-Racie,L.A.,Follettie,M.T.,Williamson,M.J.,more diverse in composition and properties than nucleicand McCoy,J.M.(1996)Histidine patch thioredoxins.Mutantforms of thioredoxin with metal chelating affinity that provide acids.In many ways,this makes them more interestingfor convenient purifications of thioredoxin fusion proteins.J. but also less amenable to generic methods and technolo-Biol.Chem.271,5059–5065.gies for parallel processing.Nonetheless,methods and10.Cronan,J.E.(1990)Biotination of proteins in vivo.A post-trans-instrumentation are currently available to meet this lational modification to label,purify,and study proteins.J.Biol.ing these advances will allow a systematic Chem.265,10327–33.effort at understanding biological pathways at the11.Maina,C.V.,Riggs,P.D.,Grandea,A.G.,Slatko,B.E.,Moran,molecular level.L.S.,Tagliamonte,J.A.,McReynolds,L.A.,and Guan,C.D.(1988)An Escherichia coli vector to express and purify foreign ACKNOWLEDGMENTS proteins by fusion to and separation from maltose-binding pro-tein.Gene74,365–373.The author acknowledges the help of Marc Nasoff,Heath Klock,Dan McMullan,Tanya Shin,Juli Vincent,Mike Hornsby,Mark12.Chong S.,Mersha F.B.,Comb D.G.,Scott M.E.,Landry D.,Vence L.M.,Perler F.B.,Benner J.,Kucera R.B.,Hirvonen C. Knuth,Loren Miraglia,and Jeremiah Gilmore for their contributionsto the high-throughput cloning and expression efforts.He also recog- A.,Pelletier J.J.,Paulus H.,and Xu M.Q.(1997)Single-column nizes Bob Downs,Mark Weselak,Andy Meyer,and Jim Mainquistpurification of free recombinant proteins using a self-cleavable and the rest of the GNF engineering staff for their contributions to affinity tag derived from a protein splicing element.Gene192, the custom robotics that make this effort possible.271–281.。
蛋白纯化(his标签)说明书
Instruction ManualProBond TM Purification SystemFor purification of polyhistidine-containing recombinant proteinsCatalog nos. K850-01, K851-01, K852-01, K853-01, K854-01,R801-01, R801-15Version K2 September200425-0006iiTable of ContentsKit Contents and Storage (iv)Accessory Products (vi)Introduction (1)Overview (1)Methods (2)Preparing Cell Lysates (2)Purification Procedure—Native Conditions (7)Purification Procedure—Denaturing Conditions (11)Purification Procedure—Hybrid Conditions (13)Troubleshooting (15)Appendix (17)Additional Protocols (17)Recipes (18)Frequently Asked Questions (21)References (22)Technical Service (23)iiiKit Contents and StorageTypes of Products This manual is supplied with the following products:Product CatalogNo.ProBond™ Purification System K850-01ProBond™ Purification System with Antibodywith Anti-Xpress™ Antibody K851-01with Anti-myc-HRP Antibody K852-01with Anti-His(C-term)-HRP Antibody K853-01with Anti-V5-HRP Antibody K854-01ProBond™ Nickel-Chelating Resin (50 ml) R801-01ProBond™ Nickel Chelating Resin (150 ml) R801-15ProBond™Purification System Components The ProBond™ Purification System includes enough resin, reagents, and columns for six purifications. The components are listed below. See next page for resin specifications.Component Composition Quantity ProBond™ Resin 50% slurry in 20% ethanol 12 ml5X NativePurification Buffer250 mM NaH2PO4, pH 8.02.5 M NaCl1 × 125 ml bottleGuanidinium LysisBuffer6 M Guanidine HCl20 mM sodium phosphate, pH 7.8500 mM NaCl1 × 60 ml bottleDenaturingBinding Buffer8 M Urea20 mM sodium phosphate, pH 7.8500 mM NaCl2 × 125 ml bottlesDenaturing WashBuffer8 M Urea20 mM sodium phosphate, pH 6.0500 mM NaCl2 × 125 ml bottlesDenaturing ElutionBuffer8 M Urea20 mM NaH2PO4, pH 4.0500 mM NaCl1 × 60 ml bottle3 M Imidazole,20 mM sodium phosphate, pH 6.0500 mM NaCl1 × 8 ml bottlePurificationColumns10 ml columns 6Continued on next pageivKit Contents and Storage, ContinuedProBond™Purification System with Antibody The ProBond™ Purification System with Antibody includes resin, reagents, and columns as described for the ProBond™ Purification System (previous page) and 50 µl of the appropriate purified mouse monoclonal antibody. Sufficient reagents are included to perform six purifications and 25 Western blots with the antibody.For more details on the antibody specificity, subclass, and protocols for using the antibody, refer to the antibody manual supplied with the system.Storage Store ProBond™ resin at +4°C. Store buffer and columns at room temperature.Store the antibody at 4°C. Avoid repeated freezing and thawing of theantibody as it may result in loss of activity.The product is guaranteed for 6 months when stored properly.All native purification buffers are prepared from the 5X Native PurificationBuffer and the 3 M Imidazole, as described on page 7.The Denaturing Wash Buffer pH 5.3 is prepared from the Denaturing WashBuffer (pH 6.0), as described on page 11.Resin and ColumnSpecificationsProBond™ resin is precharged with Ni2+ ions and appears blue in color. It isprovided as a 50% slurry in 20% ethanol.ProBond™ resin and purification columns have the following specifications:• Binding capacity of ProBond™ resin: 1–5 mg of protein per ml of resin• Average bead size: 45–165 microns• Pore size of purification columns: 30–35 microns• Recommended flow rate: 0.5 ml/min• Maximum flow rate: 2 ml/min• Maximum linear flow rate: 700 cm/h• Column material: Polypropylene• pH stability (long term): pH 3–13• pH stability (short term): pH 2–14ProductQualificationThe ProBond™ Purification System is qualified by purifying 2 mg of myoglobinprotein on a column and performing a Bradford assay. Protein recovery mustbe 75% or higher.vAccessory ProductsAdditionalProductsThe following products are also available for order from Invitrogen:Product QuantityCatalogNo.ProBond™ Nickel-Chelating Resin 50 ml150 mlR801-01R801-15Polypropylene columns(empty)50 R640-50Ni-NTA Agarose 10 ml25 ml R901-01 R901-15Ni-NTA Purification System 6 purifications K950-01 Ni-NTA Purification Systemwith Antibodywith Anti-Xpress™ Antibody with Anti-myc-HRP Antibody with Anti-His(C-term)-HRP Antibodywith Anti-V5-HRP Antibody 1 kit1 kit1 kit1 kitK951-01K952-01K953-01K954-01Anti-myc Antibody 50 µl R950-25 Anti-V5 Antibody 50 µl R960-25 Anti-Xpress™ Antibody 50 µl R910-25 Anti-His(C-term) Antibody 50 µl R930-25 InVision™ His-tag In-gel Stain 500 ml LC6030 InVision™ His-tag In-gelStaining Kit1 kit LC6033Pre-Cast Gels and Pre-made Buffers A large variety of pre-cast gels for SDS-PAGE and pre-made buffers for your convenience are available from Invitrogen. For details, visit our web site at or contact Technical Service (page 23).viIntroductionOverviewIntroduction The ProBond™ Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. Thesystem is designed around the high affinity and selectivity of ProBond™Nickel-Chelating Resin for recombinant fusion proteins containing six tandemhistidine residues.The ProBond™ Purification System is a complete system that includespurification buffers and resin for purifying proteins under native, denaturing,or hybrid conditions. The resulting proteins are ready for use in many targetapplications.This manual is designed to provide generic protocols that can be adapted foryour particular proteins. The optimal purification parameters will vary witheach protein being purified.ProBond™ Nickel-Chelating Resin ProBond™ Nickel-Chelating Resin is used for purification of recombinant proteins expressed in bacteria, insect, and mammalian cells from any 6xHis-tagged vector. ProBond™ Nickel-Chelating Resin exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins.Proteins can be purified under native, denaturing, or hybrid conditions using the ProBond™ Nickel-Chelating Resin. Proteins bound to the resin are eluted with low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.Binding Characteristics ProBond™ Nickel-Chelating Resin uses the chelating ligand iminodiacetic acid (IDA) in a highly cross-linked agarose matrix. IDA binds Ni2+ ions by three coordination sites.The protocols provided in this manual are generic, and may not result in 100%pure protein. These protocols should be optimized based on the bindingcharacteristics of your particular proteins.Native VersusDenaturingConditionsThe decision to purify your 6xHis-tagged fusion proteins under native ordenaturing conditions depends on the solubility of the protein and the need toretain biological activity for downstream applications.• Use native conditions if your protein is soluble (in the supernatant afterlysis) and you want to preserve protein activity.• Use denaturing conditions if the protein is insoluble (in the pellet afterlysis) or if your downstream application does not depend on proteinactivity.• Use hybrid protocol if your protein is insoluble but you want to preserveprotein activity. Using this protocol, you prepare the lysate and columnsunder denaturing conditions and then use native buffers during the washand elution steps to refold the protein. Note that this protocol may notrestore activity for all proteins. See page 14.1MethodsPreparing Cell LysatesIntroduction Instructions for preparing lysates from bacteria, insect, and mammalian cellsusing native or denaturing conditions are described below.Materials Needed You will need the following items:• Native Binding Buffer (recipe is on page 8) for preparing lysates undernative conditions• Sonicator• 10 µg/ml RNase and 5 µg/ml DNase I (optional)• Guanidinium Lysis Buffer (supplied with the system) for preparing lysatesunder denaturing conditions• 18-gauge needle• Centrifuge• Sterile, distilled water• SDS-PAGE sample buffer• Lysozyme for preparing bacterial cell lysates• Bestatin or Leupeptin, for preparing mammalian cell lysatesProcessing Higher Amount of Starting Material Instructions for preparing lysates from specific amount of starting material (bacteria, insect, and mammalian cells) and purification with 2 ml resin under native or denaturing conditions are described in this manual.If you wish to purify your protein of interest from higher amounts of starting material, you may need to optimize the lysis protocol and purification conditions (amount of resin used for binding). The optimization depends on the expected yield of your protein and amount of resin to use for purification. Perform a pilot experiment to optimize the purification conditions and then based on the pilot experiment results, scale-up accordingly.Continued on next page2Preparing Bacterial Cell Lysate—Native Conditions Follow the procedure below to prepare bacterial cell lysate under native conditions. Scale up or down as necessary.1. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for5 minutes in a Sorvall SS-34 rotor). Resuspend the cells in 8 ml NativeBinding Buffer (recipe on page 8).2. Add 8 mg lysozyme and incubate on ice for 30 minutes.3. Using a sonicator equipped with a microtip, sonicate the solution on iceusing six 10-second bursts at high intensity with a 10-second coolingperiod between each burst.Alternatively, sonicate the solution on ice using two or three 10-secondbursts at medium intensity, then flash freeze the lysate in liquid nitrogen or a methanol dry ice slurry. Quickly thaw the lysate at 37°C andperform two more rapid sonicate-freeze-thaw cycles.4. Optional: If the lysate is very viscous, add RNase A (10 µg/ml) andDNase I (5 µg/ml) and incubate on ice for 10–15 minutes. Alternatively,draw the lysate through a 18-gauge syringe needle several times.5. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.Note: Some 6xHis-tagged protein may remain insoluble in the pellet, and can be recovered by preparing a denatured lysate (page 4) followed bythe denaturing purification protocol (page 12). To recover this insolubleprotein while preserving its biological activity, you can prepare thedenatured lysate and then follow the hybrid protocol on page 14. Notethat the hybrid protocol may not restore activity in all cases, and should be tested with your particular protein.6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20°C. When ready to use, proceed to theprotocol on page 7.Continued on next page3Preparing Bacterial Cell Lysate—Denaturing Conditions Follow the procedure below to prepare bacterial cell lysate under denaturing conditions:1. Equilibrate the Guanidinium Lysis Buffer, pH 7.8 (supplied with thesystem or see page 19 for recipe) to 37°C.2. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for5 minutes in a Sorvall SS-34 rotor).3. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer from Step 1.4. Slowly rock the cells for 5–10 minutes at room temperature to ensurethorough cell lysis.5. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.6. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris.Transfer the supernatant to a fresh tube.7. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or at -20°C. When ready to use, proceed to the denaturingprotocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Harvesting Insect Cells For detailed protocols dealing with insect cell expression, consult the manual for your particular system. The following lysate protocols are for baculovirus-infected cells and are intended to be highly generic. They should be optimized for your cell lines.For baculovirus-infected insect cells, when the time point of maximal expression has been determined, large scale protein expression can be carried out. Generally, the large-scale expression is performed in 1 liter flasks seeded with cells at a density of 2 × 106 cells/ml in a total volume of 500 ml and infected with high titer viral stock at an MOI of 10 pfu/cell. At the point of maximal expression, harvest cells in 50 ml aliquots. Pellet the cells by centrifugation and store at -70°C until needed. Proceed to preparing cell lysates using native or denaturing conditions as described on the next page.Continued on next page4Preparing Insect Cell Lysate—Native Condition 1. Prepare 8 ml Native Binding Buffer (recipe on page 8) containingLeupeptin (a protease inhibitor) at a concentration of 0.5 µg/ml.2. After harvesting the cells (previous page), resuspend the cell pellet in8 ml Native Binding Buffer containing 0.5 µg/ml Leupeptin.3. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dryice/ethanol bath and a 42°C water bath.4. Shear DNA by passing the preparation through an 18-gauge needle fourtimes.5. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris.Transfer the supernatant to a fresh tube.6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysateon ice or freeze at -20°C. When ready to use, proceed to the protocol on page 7.Preparing Insect Cell Lysate—Denaturing Condition 1. After harvesting insect cells (previous page), resuspend the cell pellet in8 ml Guanidinium Lysis Buffer (supplied with the system or see page 19for recipe).2. Pass the preparation through an 18-gauge needle four times.3. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysateon ice or freeze at -20° C. When ready to use, proceed to the denaturingprotocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Continued on next pagePreparing Mammalian Cell Lysate—Native Conditions For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines.To produce recombinant protein, you need between 5 x 106and 1 x 107 cells. Seed cells and grow in the appropriate medium until they are 80–90% confluent. Harvest cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.1. Resuspend the cell pellet in 8 ml of Native Binding Buffer (page 8). Theaddition of protease inhibitors such as bestatin and leupeptin may benecessary depending on the cell line and expressed protein.2. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dryice/ethanol bath and a 42°C water bath.3. Shear the DNA by passing the preparation through an 18-gauge needlefour times.4. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.5. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20° C. When ready to use, proceed to theprotocol on page 7.Preparing Mammalian Cell Lysates—Denaturing Conditions For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines.To produce recombinant protein, you need between 5 x 106and 1 x 107 cells. Seed cells and grow in the appropriate medium until they are 80–90% confluent. Harvest cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.1. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer (suppliedwith the system or see page 19 for recipe).2. Shear the DNA by passing the preparation through an 18-gauge needlefour times.3. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20° C until use. When ready to use, proceed to the denaturing protocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Purification Procedure—Native ConditionsIntroduction In the following procedure, use the prepared Native Binding Buffer, NativeWash Buffer, and Native Elution Buffer, columns, and cell lysate preparedunder native conditions. Be sure to check the pH of your buffers before starting.Buffers for Native Purification All buffers for purification under native conditions are prepared from the5X Native Purification Buffer supplied with the system. Dilute and adjust the pH of the 5X Native Purification Buffer to create 1X Native Purification Buffer (page 8). From this, you can create the following buffers:• Native Binding Buffer• Native Wash Buffer• Native Elution BufferThe recipes described in this section will create sufficient buffers to perform one native purification using one kit-supplied purification column. Scale up accordingly.If you are preparing your own buffers, see page 18 for recipe.Materials Needed You will need the following items:• 5X Native Purification Buffer (supplied with the system or see page 18 forrecipe)• 3 M Imidazole (supplied with the system or see page 18 for recipe)• NaOH• HCl• Sterile distilled water• Prepared ProBond™ columns with native buffers (next page)• Lysate prepared under native conditions (page 2)Imidazole Concentration in Native Buffers Imidazole is included in the Native Wash and Elution Buffers to minimize the binding of untagged, contaminating proteins and increase the purity of the target protein with fewer wash steps. Note that, if your level of contaminating proteins is high, you may add imidazole to the Native Binding Buffer.If your protein does not bind well under these conditions, you can experiment with lowering or eliminating the imidazole in the buffers and increasing the number of wash and elution steps.Continued on next page1X Native Purification Buffer To prepare 100 ml 1X Native Purification Buffer, combine:• 80 ml of sterile distilled water• 20 ml of 5X Native Purification Buffer (supplied with the system or see page 18 for recipe)Mix well and adjust pH to 8.0 with NaOH or HCl.Native Binding Buffer Without ImidazoleUse 30 ml of the 1X Native Purification Buffer (see above for recipe) for use as the Native Binding Buffer (used for column preparation, cell lysis, and binding).With Imidazole (Optional):You can prepare the Native Binding Buffer with imidazole to reduce the binding of contaminating proteins. (Note that some His-tagged proteins may not bind under these conditions.).To prepare 30 ml Native Binding Buffer with 10 mM imidazole, combine: • 30 ml of 1X Native Purification Buffer• 100 µl of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Native Wash Buffer To prepare 50 ml Native Wash Buffer with 20 mM imidazole, combine:• 50 ml of 1X Native Purification Buffer• 335 µl of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Native Elution Buffer To prepare 15 ml Native Elution Buffer with 250 mM imidazole, combine:• 13.75 ml of 1X Native Purification Buffer• 1.25 ml of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Continued on next pageDo not use strong reducing agents such as DTT with ProBond™ columns. DTTreduces the nickel ions in the resin. In addition, do not use strong chelatingagents such as EDTA or EGTA in the loading buffers or wash buffers, as thesewill strip the nickel from the columns.Be sure to check the pH of your buffers before starting.PreparingProBond™ ColumnWhen preparing a column as described below, make sure that the snap-off capat the bottom of the column remains intact. To prepare a column:1. Resuspend the ProBond™ resin in its bottle by inverting and gentlytapping the bottle repeatedly.2. Pipet or pour 2 ml of the resin into a 10-ml Purification Columnsupplied with the kit. Allow the resin to settle completely by gravity(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minuteat 800 ×g). Gently aspirate the supernatant.3. Add 6 ml of sterile, distilled water and resuspend the resin byalternately inverting and gently tapping the column.4. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.5. For purification under Native Conditions, add 6 ml Native BindingBuffer (recipe on page 8).6. Resuspend the resin by alternately inverting and gently tapping thecolumn.7. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.8. Repeat Steps 5 through 7.Storing PreparedColumnsTo store a column containing resin, add 0.02% azide or 20% ethanol as apreservative and cap or parafilm the column. Store at room temperature.Continued on next pagePurification Under Native Conditions Using the native buffers, columns and cell lysate, follow the procedure below to purify proteins under native conditions:1. Add 8 ml of lysate prepared under native conditions to a preparedPurification Column (page 9).2. Bind for 30–60 minutes using gentle agitation to keep the resinsuspended in the lysate solution.3. Settle the resin by gravity or low speed centrifugation (800 ×g), andcarefully aspirate the supernatant. Save supernatant at 4°C forSDS-PAGE analysis.4. Wash with 8 ml Native Wash Buffer (page 8). Settle the resin by gravityor low speed centrifugation (800 ×g), and carefully aspirate thesupernatant. Save supernatant at 4°C for SDS-PAGE analysis.5. Repeat Step 4 three more times.6. Clamp the column in a vertical position and snap off the cap on thelower end. Elute the protein with 8–12 ml Native Elution Buffer (seepage 2). Collect 1 ml fractions and analyze with SDS-PAGE.Note: Store the eluted fractions at 4°C. If -20°C storage is required, addglycerol to the fractions. For long term storage, add protease inhibitors to the fractions.If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. If you need to recharge the resin, see page 17.Purification Procedure—Denaturing ConditionsIntroduction Instructions to perform purification using denaturing conditions with prepareddenaturing buffers, columns, and cell lysate are described below.Materials Needed You will need the following items:• Denaturing Binding Buffer (supplied with the system or see page 19 forrecipe)• Denaturing Wash Buffer, pH 6.0 (supplied with the system or see page 19 forrecipe) and Denaturing Wash Buffer, pH 5.3 (see recipe below)• Denaturing Elution Buffer (supplied with the system or see page 20 forrecipe)• Prepared ProBond™ columns with Denaturing buffers (see below)• Lysate prepared under denaturing conditions (page 11)Preparing the Denaturing Wash Buffer pH 5.3 Using a 10 ml aliquot of the kit-supplied Denaturing Wash Buffer (pH 6.0), mix well, and adjust the pH to 5.3 using HCl. Use this for the Denaturing Wash Buffer pH 5.3 in Step 5 next page.Be sure to check the pH of your buffers before starting. Note that thedenaturing buffers containing urea will become more basic over time. PreparingProBond™ ColumnWhen preparing a column as described below, make sure that the snap-off capat the bottom of the column remains intact.If you are reusing the ProBond™ resin, see page 17 for recharging protocol.To prepare a column:1. Resuspend the ProBond™ resin in its bottle by inverting and gentlytapping the bottle repeatedly.2. Pipet or pour 2 ml of the resin into a 10-ml Purification Columnsupplied with the kit. Allow the resin to settle completely by gravity(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minuteat 800 ×g). Gently aspirate the supernatant.3. Add 6 ml of sterile, distilled water and resuspend the resin byalternately inverting and gently tapping the column.4. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.5. For purification under Denaturing Conditions, add 6 ml of DenaturingBinding Buffer.6. Resuspend the resin by alternately inverting and gently tapping thecolumn.7. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.Continued on next pagePurification Procedure—Denaturing Conditions, ContinuedPurification Under Denaturing Conditions Using the denaturing buffers, columns, and cell lysate, follow the procedure below to purify proteins under denaturing conditions:1. Add 8 ml lysate prepared under denaturing conditions to a preparedPurification Column (page 11).2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g.,using a rotating wheel) to keep the resin suspended in the lysatesolution. Settle the resin by gravity or low speed centrifugation (800 ×g), and carefully aspirate the supernatant.3. Wash the column with 4 ml Denaturing Binding Buffer supplied with thekit by resuspending the resin and rocking for two minutes. Settle theresin by gravity or low speed centrifugation (800 ×g), and carefullyaspirate the supernatant. Save supernatant at 4°C for SDS-PAGEanalysis. Repeat this step one more time.4. Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 supplied inthe kit by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 ×g), and carefullyaspirate the supernatant. Save supernatant at 4°C for SDS-PAGEanalysis. Repeat this step one more time.5. Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 (see recipeon previous page) by resuspending the resin and rocking for 2 minutes.Settle the resin by gravity or low speed centrifugation (800 ×g), andcarefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis. Repeat this step once more for a total of two washes with Denaturing Wash Buffer pH 5.3.6. Clamp the column in a vertical position and snap off the cap on thelower end. Elute the protein by adding 5 ml Denaturing Elution Buffersupplied with the kit. Collect 1 ml fractions and monitor the elution bytaking OD280readings of the fractions. Pool the fractions that contain the peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X-100 overnight at 4°C to remove the urea. Concentrate the dialyzedmaterial by any standard method (i.e., using 10,000 MW cut-off, low-protein binding centrifugal instruments or vacuum concentrationinstruments).If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. If you need to recharge the resin, see page 17.。
Protein purification
Electrophoresis is largely used as an analytical tool - mostly to answer is your protein pure? It is rarely used as a is an analytical technique
5
Nickel Chromatography (IMAC - immobilized metal affinity chromatography)
• A specific kind of affinity chromatography • Protein is non-native - relies on a ‘tag’
• Electrophoresis
We need pure proteins!
• Our determination of protein structure and function requires pure proteins.
– Crystallization – Interaction studies
SDS-gels of a purification in the Macbeth Lab
A histidine tagged RNA editing protein. Binds tightly to an RNA (which type of ion exchange?) fractions from each column are run on an SDS-gel
Protein Purification
• Native or Overexpressed? • Cellular Fractionation • Chromatography
– – – – – – – – Ion exchange Size exclusion Affinity Hydrophobic interaction Native SDS 2-D electrophoresis Isoelectric focusing
protein purification and characterization
Protein production may be from:- Extra cellular - Intra cellular Protein production by Recombinant DNA technology:- Homologous - Heterologous. * Plants * Animals (tissues)
Protein Purification and Characaman
Contact Information
Department of Biological Sciences S3 Level 04-08 Tel: 6874 1163 Fax: 6779 5671 E-mail: dbsjayar@.sg
Preliminary Purification/Conc.
Commonly used methods are :
Dialysis using cellulose tubing Concentration by precipitation (salt, solvent etc.), Gel filtration, Ion-exchange chromatography etc.. Ultra-filtration through membrane system e.g. Centriprep MWCO 3000 / 10000 Lyophilization
Organic Precipitation
Ordered water molecule structure around hydrophobic areas displaced by organic solvent molecules (higher solubility of these molecules) Aggregation and precipitation occur due to electrostatic and dipolar van der waals forces Similarly for organic polymers (like PEG)
5蛋白组学定量蛋白质组学
d0/d4标记蛋白质N末端原理
HH H
H
HH
H
2
H
2
H4-Nic-NHS[1-(H4-烟酰氧基)琥珀酰亚胺] 四氘代 H4-Nic-NHS[1-(H4-烟酰氧基)琥珀酰亚胺]
23
MALDI-TOF mass spectrum of a peptide labeled with
succinic anhydride and deuterated succinic anhydride
36
–COOH羧基标记
通过对羧基酯化进行 标记
用H和D标记的甲醇酯 化标记，来定量研究 蛋白质表达量的差异
37
羧基酯化标记进行蛋白定量研究
比例＝2 : 1
38
缺点：
特异性不是很好，在C末端和Asp和 Glu残基上都有标记，且效率不均
采用的标记条件容易引起天冬酰胺 (Asn)和谷氨酰胺(Gln)的去酰胺
通过离子交换层析图 谱上峰的面积，可以 计算两种肽段的相对 量 0.88 : 1
35
MCAT优点
O-甲基异脲(O-methylisourea)对Lys残基εNH2的胍基化程度可以进行量上的控制
增加的质量（每个胍基42 Da）可以很容 易在MS上检测到
ε-NH2的胍基化修饰并不影响肽段的离子 化和带电荷程度
31
MCAT策略流程：定性
Lys只在Trypsin酶切后的末端，所以产生的b离子都没有被修饰；所有的y 离子带Lys，因此被修饰。
在MS/MS图谱上，所有的b离子是单一条带出现，所有的y离子是成对出现， 丰度比例一样，且相差同样的m/z。
容易区分b离子和y离子，容易读出氨基酸序列。
32
MCAT策略流程：定量
纯化技术-Purification
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
© UF
© UF
GFP Purification Procedures
Day 1 Day 2
© UF
Day 3
/LifeScience/docs/Official_pGLO_GFP_powerpoint_Spring_2005.ppt
Hydrophobic Interaction Chromatography: Steps 1–3
Hydrophobic bead
© UF
Step 1: Hydrophobic Interaction Chromatography
• Add bacterial lysate to column matrix in high salt buffer
– Hydrophobic proteins interact with column
– Less hydrophobic E. coli proteins fall from column – GFP remains bound to the column
© UF
/LifeScience/docs/Official_pGLO_GFP_powerpoint_Spring_2005.ppt
Process Development
1. Identification of target protein w/therapeutic value 2. Identification of target gene 3. Isolation of the target gene 4. Insertion of the target gene into a host cell (such as E.coli) & express protein 5. Purification: Separation of the target protein from the host cell protein
蛋白质分离技术
★等电点测定
等电聚焦法（isoelectricfocusing）
（2）蛋白质的胶体性质
蛋白质的分子量很大（1-100nm），在水溶液中 具有胶体性质，如布郎运动、丁达尔现象不能 通过半透膜等。 透析：将含小分子杂质的蛋白质放入透析袋，置 水中，小分子杂质不断从袋中出来，大分子蛋 白质仍留在袋中。 半透膜：只允许小分子通过，而大分子不能通过。 如羊皮纸、火棉胶、玻璃纸等。
等电聚焦法
2、离子交换层析法
基质：纤维素、交联葡聚糖、树脂
电荷基团：
（四） 配体亲和力的差异 ： 亲和层析
亲和层析:利用蛋白质分子对其配体分子特有的 识别能力。 配基：
酶的底物、辅酶、抑制剂、效应物及其结构类似 物，激素与受体蛋白，抗原与抗体，生物素与 亲和素（抗亲和素蛋白），凝集素。
（五）HPLC（high performance/pressure liquid chromatography)
第四节：蛋白质的重要性质及分离纯化
Release proteins from cells and Differential centrifugation as a first step in protein purification
一、蛋白质的性质
（1）蛋白质的两性解离
蛋白质与aa一样，能够发生两性解离， 也有等电点。在等电点时(Isoelectric point pI)，蛋白质的溶解度最小，在 电场中不移动。
（5）蛋白质的紫外吸收
Trp、Tyr和Phe在280nm 附近有最大吸收。 因此，利用这个性质，可以对蛋白质进 行定性鉴定和定量测定。
二、分离纯化蛋白质的主要方法
（一）根据溶解度差异分离：选择性沉淀 法
蛋白质纯化手册【ProteinPurificationHandbook】
Protein PurificationHandbook18-1132-29Edition ABHiTrap, Sepharose, STREAMLINE, Sephadex, MonoBeads, Mono Q,Mono S, MiniBeads, RESOURCE, SOURCE, Superdex, Superose, HisTrap, HiLoad, HiPrep, INdEX, BPG, BioProcess, FineLINE, MabTrap, MAbAssistant, Multiphor, FPLC, PhastSystem and ÄKTA are trademarks of Amersham Pharmacia Biotech Limitedor its subsidiaries.Amersham is a trademark of Nycomed Amersham plcPharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc Coomassie is a trademark of ICI plcAll goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions of sale is available on request.© Amersham Pharmacia Biotech AB 1999-All rights reserved.Amersham Pharmacia Biotech ABSE-751 84 Uppsala SwedenAmersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech Inc800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USAProtein Purification HandbookContents Introduction (7)Chapter 1Purification Strategies - A Simple Approach (9)Preparation (10)Three Phase Purification Strategy (10)General Guidelines for Protein Purification (12)Chapter 2 Preparation (13)Before You Start (13)Sample Extraction and Clarification (16)Chapter 3Three Phase Purification Strategy (19)Principles (19)Selection and Combination of Purification Techniques (20)Sample Conditioning (26)Chapter 4Capture (29)Chapter 5Intermediate Purification (37)Chapter 6Polishing (40)Chapter 7Examples of Protein Purification Strategies (45)Three step purification of a recombinant enzyme (45)Three step purification of a recombinant antigen binding fragment (49)Two step purification of a monoclonal antibody (54)One step purification of an integral membrane protein (57)Chapter 8Storage Conditions (61)Extraction and Clarification Procedures (62)Chapter 9Principles and Standard Conditions for Purification Techniques (73)Ion exchange (IEX) (73)Hydrophobic interaction (HIC) (79)Affinity (AC) (85)Gel filtration (GF) (88)Reversed phase (RPC) (92)Expanded bed adsorption (EBA) (95)IntroductionThe development of techniques and methods for protein purification has been an essential pre-requisite for many of the advancements made in biotechnology. This booklet provides advice and examples for a smooth path to protein purification. Protein purification varies from simple one-step precipitation procedures to large scale validated production processes. Often more than one purification step is necessary to reach the desired purity. The key to successful and efficient protein purification is to select the most appropriate techniques, optimise their performance to suit the requirements and combine them in a logical way to maximise yield and minimise the number of steps required.Most purification schemes involve some form of chromatography. As a result chromatography has become an essential tool in every laboratory where protein purification is needed. The availability of different chromatography techniques with different selectivities provides a powerful combination for the purification of any biomolecule.Recombinant DNA developments over the past decade have revolutionised the production of proteins in large quantities. Proteins can even be produced in forms which facilitate their subsequent chromatographic purification. However, this has not removed all challenges. Host contaminants are still present and problems related to solubility, structural integrity and biological activity can still exist. Although there may appear to be a great number of parameters to consider, with a few simple guidelines and application of the Three Phase Purification Strategy the process can be planned and performed simply and easily, with only a basic knowledge of the details of chromatography techniques.78Chapter 1Purification Strategies- a simple approachApply a systematic approach to development of a purification strategy. The first step is to describe the basic scenario for the purification. General considerations answer questions such as: What is the intended use of the product? What kind of starting material is available and how should it be handled? What are the purity issues in relation to the source material and intended use of the final product? What has to be removed? What must be removed completely? What will be the final scale of purification? If there is a need for scale-up, what consequences will this have on the chosen purification techniques? What are the economical constraints and what resources and equipment are available?Most purification protocols require more than one step to achieve the desired level of product purity. This includes any conditioning steps necessary to transfer the product from one technique into conditions suitable to perform the next technique. Each step in the process will cause some loss of product. For example, if a yield of 80% in each step is assumed, this will be reduced to only 20% overall yield after 8 processing steps as shown in Figure 1. Consequently, to reach the targets for yield and purity with the minimum number of steps and the simplest possible design, it is not efficient to add one step to another until purity requirements have been fulfilled. Occasionally when a sample is readily available purity can be achieved by simply adding or repeating steps. However, experience shows that, even for the most challenging applications, high purity and yield can be achieved efficiently in fewer than four well-chosen and optimised purification steps. Techniques should be organised in a logical sequence to avoid the need for conditioning steps and the chromatographic techniques selected appropriately to use as few purification steps as possible.Limit the number of steps in a purification procedure910Fig.1.Yields from multi-step purifications.PreparationThe need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to every purification. It is important to set objectives for purity,quantity and maintenance of biological activity and to define the economical and time framework for the work. All information concerning properties of the target protein and contaminants will help during purification development. Some simple experiments to characterise the sample and target molecule are an excellent investment. Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess its effectiveness. Sample preparation and extraction procedures should be developed prior to the first chromatographic purification step.With background information, assays and sample preparation procedures in place the Three Phase Purification Strategy can be considered.Three Phase Purification Strategy Imagine the purification has three phases Capture, IntermediatePurification and Polishing.In the Three Phase Strategy specific objectives are assigned to each step within the process:In the capture phase the objectives are to isolate, concentrate and stabilise the target product.During the intermediate purification phase the objective is to remove most of the bulk impurities such as other proteins and nucleic acids, endotoxins and viruses.In the polishing phase the objective is to achieve high purity by removing any remaining trace impurities or closely related substances.The selection and optimum combination of purification techniques for Capture,Intermediate Purification and Polishing is crucial to ensure fast method development, a shorter time to pure product and good economy.108060402012345678Number of steps 95% / step90% / step 85% / step 80% / step 75% / stepYield (%)The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by three major purification steps, as shown in Figure 2. The number of steps used will always depend upon the purity requirements and intended use for the protein.Fig. 2.Preparation and the Three Phase Purification Strategy11Guidelines for Protein PurificationThe guidelines for protein purification shown here can be applied to any purification process and are a suggestion as to how a systematic approach can be applied to the development of an effective purification strategy. As a reminder these guidelines will be highlighted where appropriate throughout the following chapters.Define objectivesfor purity, activity and quantity required of final product to avoid over or under developing a methodDefine properties of target protein and critical impuritiesto simplify technique selection and optimisationDevelop analytical assaysfor fast detection of protein activity/recovery and to work efficientlyMinimise sample handling at every stageto avoid lengthy procedures which risk losing activity/reducing recovery Minimise use of additivesadditives may need to be removed in an extra purification step or may interfere with activity assaysRemove damaging contaminants earlyfor example, proteasesUse a different technique at each stepto take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity)Minimise number of stepsextra steps reduce yield and increase time, combine steps logicallyKEEP IT SIMPLE!12Chapter 2PreparationBefore You StartThe need to obtain a protein, efficiently, economically and in sufficient purity and quantity, applies to any purification, from preparation of an enriched protein extract for biochemical characterisation to large scale production of a therapeutic recombinant protein. It is important to set objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time. Purity requirements must take into consideration the nature of the source material, the intended use of the final product and any special safety issues. For example, it is important to differentiate between contaminants which must be removed and those which can be tolerated. Other factors can also influence the prioritisation of objectives. High yields are usually a key objective, but may be less crucial in cases where a sample is readily available or product is required only in small quantities. Extensive method development may be impossible without resources such as an ÄKTA™design chromatography system. Similarly, time pressure combined with a slow assay turnaround will steer towards less extensive scouting and optimisation. All information concerning properties of the target protein and contaminants will help during purification development, allowing faster and easier technique selection and optimisation, and avoiding conditions which may inactivate the target protein.Development of fast and reliable analytical assays is essential to follow the progress of the purification and assess effectiveness (yield, biological activity, recovery).Define objectivesGoal:To set minimum objectives for purity and quantity, maintenance of biological activity and economy in terms of money and time.Define purity requirements according to the final use of the product. Purity requirement examples are shown below.Extremely high > 99%Therapeutic use, in vivo studiesHigh 95- 99 %X-ray crystallography and most physico-chemicalcharacterisation methodsModerate < 95 %Antigen for antibody productionN-terminal sequencing13Identify 'key' contaminantsIdentify the nature of possible remaining contaminants as soon aspossible.The statement that a protein is >95% pure (i.e. target protein constitutes 95% of total protein) is far from a guarantee that the purity is sufficient for an intended application. The same is true for the common statement "the protein was homogenous by Coomassie™ stained SDS-PAGE". Purity of 95% may be acceptable if the remaining 5% consists of harmless impurities. However, even minor impurities which may be biologically active could cause significant problems in both research and therapeutic applications. It is therefore important to differentiate between contaminants which must be removed completely and those which can be reduced to acceptable levels. Since different types of starting material will contain different contaminant profiles they will present different contamination problems.It is better to over-purify than to under-purify.Although the number of purification steps should be minimised, thequality of the end product should not be compromised. Subsequent results might be questioned if sample purity is low and contaminants are unknown.Contaminants which degrade or inactivate the protein or interfere withanalyses should be removed as early as possible.The need to maintain biological activity must be considered at every stage during purification development. It is especially beneficial if proteases are removed and target protein transferred into a friendly environment during the first step.Economy is a very complex issue. In commercial production the time to market can override issues such as optimisation for recovery, capacity or speed. Robustness and reliability are also of great concern since a batch failure can have major consequences.It may be necessary to use analytical techniques targetted towards specific conta-minants in order to demonstrate that they have been removed to acceptable levels. 14Define properties of target protein and critical impurities Goal:To determine a 'stability window' for the target protein for easier selection and optimisation of techniques and to avoid protein inactivation during purification.Check target protein stability window for at least pH and ionic strength. All information concerning the target protein and contaminant properties will help to guide the choice of separation techniques and experimental conditions for purification. Database information for the target, or related proteins, may give size, isoelectric point (pI) and hydrophobicity or solubility data. Native one and two dimensional PAGE can indicate sample complexity and the properties of the target protein and major contaminants. Particularly important is a knowledge of the stability window of the protein so that irreversible inactivation is avoided. Itis advisable to check the target protein stability window for at least pH and ionic strength. Table 1 shows how different target protein properties can affect a purification strategy.Table 1.Protein properties and their effect on development of purification strategies. Sample and target protein properties Influence on purification strategyTemperature stability Need to work rapidly at lowered temperaturepH stability Selection of buffers for extraction and purificationSelection of conditions for ion exchange, affinity orreversed phase chromatographyOrganic solvents stability Selection of conditions for reversed phasechromatographyDetergent requirement Consider effects on chromatographic steps and the needfor detergent removal. Consider choice of detergent.Salt (ionic strength)Selection of conditions for precipitation techniques andhydrophobic interaction chromatographyCo-factors for stability or activity Selection of additives, pH, salts, buffersProtease sensitivity Need for fast removal of proteases or addition ofinhibitorsSensitivity to metal ions Need to add EDTA or EGTA in buffersRedox sensitivity Need to add reducing agentsMolecular weight Selection of gel filtration mediaCharge properties Selection of ion exchange conditionsBiospecific affinity Selection of ligand for affinity mediumPost translational modifications Selection of group specific affinity medium Hydrophobicity Selection of medium for hydrophobic interactionchromatography15Develop analytical assaysGoal:To follow the progress of a purification, to assess effectiveness (yield, biological activity, recovery) and to help during optimisation.Select assays which are fast and reliable.To progress efficiently during method development the effectiveness of each step should be assessed. The laboratory should have access to the following assays:• A rapid, reliable assay for the target protein• Purity determination• Total protein determination• Assays for impurities which must be removedThe importance of a reliable assay for the target protein cannot be over- emphasised. When testing chromatographic fractions ensure that the buffers used for separation do not interfere with the assay. Purity of the target protein is most often estimated by SDS-PAGE, capillary electrophoresis, reversed phase chromatography or mass spectrometry. Lowry or Bradford assays are used most frequently to determine the total protein.The Bradford assay is particularly suited to samples where there is a high lipid content which may interfere with the Lowry assay.For large scale protein purification the need to assay for target proteins and critical impurities is often essential. In practice, when a protein is purified for research purposes, it is too time consuming to identify and set up specific assays for harmful contaminants. A practical approach is to purify the protein to a certain level, and then perform SDS-PAGE after a storage period to check for protease cleavage. Suitable control experiments, included within assays forbio-activity, will help to indicate if impurities are interfering with results.Sample Extraction and Clarification Minimise sample handlingMinimise use of additivesRemove damaging contaminants earlyDefinition:Primary isolation of target protein from source material.Goal:Preparation of a clarified sample for further purification. Removal of particulate matter or other contaminants which are not compatible with chromatography.16The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose™ Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure.If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used. It should be noted that a technique such as ammonium sulphate precipitation, commonly used in small scale, may be unsuitable for very large scale preparation. Choice of buffers and additives must be carefully considered if a purification is to be scaled up. In these cases inexpensive buffers, such as acetate or citrate, are preferable to the more complex compositions used in the laboratory. It should also be noted that dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples.For repeated purification, use an extraction and clarification techniquethat is robust and able to handle sample variability. This ensures areproducible product for the next purification step despite variability instarting material.Use additives only if essential for stabilisation of product or improvedextraction. Select those which are easily removed. Additives may need tobe removed in an extra purification step.Use pre-packed columns of Sephadex™ G-25 gel filtration media, forrapid sample clean-up at laboratory scale, as shown in Table 2.Table 2.Pre-packed columns for sample clean-up.Pre-packed column Sample volume Sample volume Code No.loading per run recovery per runHiPrep™Desalting 26/10 2.5 -15 ml7.5 - 20 ml17-5087-01HiTrap Desalting0.25 - 1.5 ml 1.0 - 2.0 ml17-1408-01Fast Desalting PC 3.2/100.05 - 0.2 ml0.2 - 0.3 ml17-0774-01PD-10 Desalting 1.5 - 2.5 ml 2.5 - 3.5 ml17-0851-01 Sephadex G-25 gel filtration media are used at laboratory and production scale for sample preparation and clarification of proteins >5000. Sample volumes of up to 30%, or in some cases, 40% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity, relative insensitivity to sample concentration, and speed of this step enable very large sample volumes to be processed rapidly and efficiently. Using a high sample volume load results in a separation with minimal sample dilution (approximately 1:1.4). Chapter 8 contains further details on sample storage, extraction and clarification procedures.17Sephadex G-25 is also used for sample conditioning i.e. rapid adjustment of pH, buffer exchange and desalting between purification steps.Sephadex G-25 gel filtrationFor fast group separations between high and low molecular weight substances Typical flow velocity 60 cm/h (Sephadex G-25 SuperFine, Sephadex G-25 Fine), 150 cm/h (Sephadex G-25 Medium).If large sample volumes will be handled or the method scaled-up in the future, consider using STREAMLINE™ expanded bed adsorption. This technique is particularly suited for large scale recombinant protein and monoclonal antibody purification. The crude sample containing particles can be applied to the expanded bed without filtration or centrifugation. STREAMLINE adsorbents are specially designed for use in STREAMLINE columns. Together they enable the high flow rates needed for high productivity in industrial applications of fluidised beds. The technique requires no sample clean up and so combines sample preparation and capture in a single step. Crude sample is applied to an expanded bed STREAMLINE media. Target proteins are captured whilst cell debris, cells, particulate matter, whole cells, and contaminants pass through. Flow is reversed and the target proteins are desorbed in the elution buffer.STREAMLINE (IEX, AC, HIC)For sample clean-up and capture direct from crude sample.STREAMLINE adsorbents are designed to handle feed directly from both fermentation homogenate and crude feedstock from cell culture/fermentation at flow velocities of 200 - 500 cm/h, according to type and application.Particle size: 200 µmNote:cm/h: flow velocity (linear flow rate) = volumetric flow rate/cross sectional area of column.18Chapter 3Three Phase Purification StrategyPrinciplesWith background information, assays, and sample preparation and extraction procedures in place the Three Phase Purification Strategy can be applied (Figure 3). This strategy is used as an aid to the development of purification processes for therapeutic proteins in the pharmaceutical industry and is equally efficient as an aid when developing purification schemes in the research laboratory.Fig. 3.Preparation and the Three Phase Purification Strategy.Assign a specific objective to each step within the purification process.In the Three Phase Strategy a specific objective is assigned to each step. The purification problem associated with a particular step will depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process i.e. at the beginning for isolation of product from crude sample, in the middle for further purification of partially purified sample, or at the end for final clean up of an almost pure product.The Three Phase Strategy ensures faster method development, a shorter time to pure product and good economy.In the capture phase the objectives are to isolate, concentrate and stabilise the target product. The product should be concentrated and transferred to an environment which will conserve potency/activity. At best, significant removal of other critical contaminants can also be achieved.19During the intermediate purification phase the objectives are to remove most of the bulk impurities,such as other proteins and nucleic acids, endotoxins and viruses.In the polishing phase most impurities have already been removed except for trace amounts or closely related substances. The objective is to achieve final purity.It should be noted that this Three Phase Strategy does not mean that all strategies must have three purification steps. For example, capture and intermediate purification may be achievable in a single step, as may intermediate purification and polishing. Similarly, purity demands may be so low that a rapid capture step is sufficient to achieve the desired result, or the purity of the starting material may be so high that only a polishing step is needed. For purification of therapeutic proteins a fourth or fifth purification step may be required to fulfil the highest purity and safety demands.The optimum selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification process.Selection and Combination ofPurification TechniquesMinimise sample handlingMinimise number of stepsUse different techniques at each stepGoal:Fastest route to a product of required purity.For any chromatographic separation each different technique will offer different performance with respect to recovery, resolution, speed and capacity. A technique can be optimised to focus on one of these parameters, for example resolution, or to achieve the best balance between two parameters, such as speed and capacity.A separation optimised for one of these parameters will produce results quite different in appearance from those produced using the same technique, but focussed on an alternative parameter. See, for example, the results shown on page 49 where ion exchange is used for a capture and for a polishing step.20Select a technique to meet the objectives for the purification step. Capacity,in the simple model shown, refers to the amount of target protein loaded during purification. In some cases the amount of sample which can be loaded may be limited by volume (as in gel filtration) or by large amounts of contaminants rather than the amount of the target protein.Speed is of the highest importance at the beginning of a purification where contaminants such as proteases must be removed as quickly as possible. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and unfavourable conditions on the column. Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix to produce narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties.Every technique offers a balance between resolution, speed, capacity and recovery and should be selected to meet the objectives for each purification step. In general, optimisation of any one of these four parameters can only be achieved at the expense of the others and a purification step will be a compromise. The importance of each parameter will vary depending on whether a purification step is used for capture, intermediate purification or polishing. This will steer the optimisation of the critical parameters, as well as the selection of the most suitable media for the step.Proteins are purified using chromatographic purification techniques which separate according to differences in specific properties, as shown in Table 3. Table 3.Protein properties used during purification.Protein property TechniqueCharge Ion exchange (IEX)Size Gel filtration (GF)Hydrophobicity Hydrophobic interaction (HIC),reversed phase (RPC)Biorecognition (ligand specificity)Affinity (AC)Charge, ligand specificity or hydrophobicity Expanded bed adsorption (EBA) follows theprinciples of AC, IEX or HIC21。
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• Mammalian Cells: Full range of modifications. Poor yield, hard to grow. High cost.
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Lyse Cells
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Prevent Proteolysis and Keep the Protein Stable
• Cold temperature
– Also inhibits growth of bacteria
• Chemicals that Inhibit Proteases
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Affinity Tags Used for Purification of Recombinant
Proteins
• GST: Glutathione-S-Transferase. Purify w/ column of glutathione. Elute w/ same.
• MBP: Maltose-Binding Protein. Purify w/ column of amylose. Elute w/ maltose.
– EDTA to inhibit Ca++ dependent proteases
• Test a variety of buffer conditions
– Find one where enzyme activity remains constant over time
精PYDVPDYA – Myc: EQKLISEEDL – FLAG: DYKDDDDK
• Purify w/ affinity beads containing a monoclonal antibody that binds tightly to the peptide
• Elute w/ peptide
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Using SDS-PAGE to Assess Purity
Separate the Polypeptides Migrate
Based on Mass
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Determining Mass of Polypeptide
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Purifying Proteins
Department of Physiology and Pathophysiolog Chao Lu
Email: luchao@
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• Preserve the structure during purification 精品课件 • Consider that the structure may be lost
No post-translational modifications. Low cost.
• Yeast: Intermediate Ease of Growth and Yield of Protein. Some modifications: Lipid, Carbohydrate. Low cost.
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Affinity-tag Purification
• Optional: Place a very specific (i.e. rate) proteolytic cleavage site between the Tag and YFP.
– TEV Protease: ENLYFQG – PreScission Protease: LEVLFQ/GP
• Express in bacteria, yeast, insect or mammalian cells
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Advantages and Disadvantages of Protein Expression Systems
• /gw_file.php?fid=6033 • Bacteria: Large amounts of protein, Easy to grow.
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Dialysis to Change Solution Conditions
• For Various Steps, often want protein mixture to be in a certain buffer
• Dialyse the sample against a semi-permeable membrane
Ammonium Sulfate Precipitation
• Very high ionic strength Proteins precipitate “Salting Out”
• Modest
Purification but
Also Useful to
Concentrate the
Sample
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Typical 2-D Gel from Whole Cell or Tissue Sample
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Affinity-tag Purification
• Use Recombinant DNA Technology to Create a Fusion Protein containing the Tag and Your Favorite Protein (YFP).
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Affinity chromatography
• 6xHis: Poly-Histidine. Purify w/ metal (Nickel or Cobalt) resins. Elute w/ imidazole or low pH.
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Antibody-based Purification
• Fuse a short Peptide to Your Favorite Protein