Protein Purification: Principles, High Resolution Methods, and Applications, Third Edition PDF
Preview Protein Purification: Principles, High Resolution Methods, and Applications, Third Edition
PROTEIN PURIFICATION PROTEIN PURIFICATION Principles, High Resolution Methods, and Applications Third Edition Editedby JAN-CHRISTER JANSON Copyright#2011byJohnWiley&Sons,Inc.Allrightsreserved PublishedbyJohnWiley&Sons,Inc.,Hoboken,NewJersey PublishedsimultaneouslyinCanada Nopartofthispublicationmaybereproduced,storedinaretrievalsystem,ortransmittedinanyformorbyanymeans,electronic,mechanical,photocopying, recording,scanning,orotherwise,exceptaspermittedunderSection107or108ofthe1976UnitedStatesCopyrightAct,withouteitherthepriorwrittenper- missionofthePublisher,orauthorizationthroughpaymentoftheappropriateper-copyfeetotheCopyrightClearanceCenter,Inc.,222RosewoodDrive,Danvers, MA01923,(978)750-8400,fax(978)750-4470,oronthewebatwww.copyright.com.RequeststothePublisherforpermissionshouldbeaddressedtothe PermissionsDepartment,JohnWiley&Sons,Inc.,111RiverStreet,Hoboken,NJ07030,(201)748-6011,fax(201)748-6008,oronlineathttp://www.wiley. com/go/permission. 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Wileyalsopublishesitsbooksinvarietyofelectronicformats.Somecontentthatappearsinprintmaynotbeavailableinelectronicformat.Formoreinformation aboutWileyproducts,visitourwebsiteatwww.wiley.com. LibraryofCongressCataloging-in-PublicationData: Proteinpurification:principles,highresolutionmethods,andapplications/editedbyJan-ChristerJanson.—3rded. p.cm. Includesindex. ISBN978-0-471-74661-4(cloth) 1. Proteins–Purification. 2. Chromatographicanalysis. 3. Electrophoresis. I. Janson,Jan-Christer. QP551.P697542011 5720.6—dc22 2010033316 PrintedintheUnitedStatesofAmerica 10 9 8 7 6 5 4 3 2 1 CONTENTS PREFACE TO THE THIRD EDITION vii PREFACE TO THE SECOND EDITION ix PREFACE TO THE FIRST EDITION xi CONTRIBUTORS xiii PART I INTRODUCTION 1 1 IntroductiontoProteinPurification 3 BoErsson,LarsRyde´n,andJan-ChristerJanson PART II CHROMATOGRAPHY 23 2 IntroductiontoChromatography 25 Jan-ChristerJansonandJanA˚keJo¨nsson 3 GelFiltration:SizeExclusionChromatography 51 LarsHagel 4 IonExchangeChromatography 93 EvertKarlssonandIrwinHirsh 5 High-ResolutionReversed-PhaseChromatographyofProteins 135 SylviaWinkelPettersson 6 HydrophobicInteractionChromatography 165 Kjell-OveErikssonandMakonnenBelew 7 ImmobilizedMetalIonAffinityChromatography 183 LennartKa˚gedal 8 CovalentChromatography 203 FranciscoBatista-Viera,LarsRyde´n,andJanCarlsson 9 AffinityChromatography 221 FranciscoBatista-Viera,Jan-ChristerJanson,andJanCarlsson v vi CONTENTS 10 AffinityLigandsfromChemicalCombinatorialLibraries 259 EnriqueCarredanoandHerbertBaumann 11 AffinityLigandsfromBiologicalCombinatorialLibraries 269 Per-A˚keNygren PART III OTHER SEPARATION METHODS AND RELATED TECHNIQUES 279 12 MembraneSeparations 281 JoachimK.Walter,ZuweiJin,MaikW.Jornitz,andUweGottschalk 13 RefoldingofInclusionBodyProteinsfromE.coli 319 ZhiguoSu,DiannanLu,andZhengLiu 14 PurificationofPEGylatedProteins 339 ConanJ.FeeandJamesM.VanAlstine PART IV ELECTROPHORESIS 363 15 ElectrophoresisinGels 365 ReinerWestermeier 16 ConventionalIsoelectricFocusinginGelSlabsandCapillariesandImmobilizedpHGradients 379 PierGiorgioRighetti,ElisaFasoli,andSabinaCarlaRighetti 17 Two-DimensionalElectrophoresisinProteomics 411 ReinerWestermeierandAngelikaGo¨rg 18 ProteinElutionandBlottingTechniques 441 ReinerWestermeier 19 CapillaryElectrophoreticSeparations 451 WolfgangThormann PART V SEPARATION METHOD OPTIMIZATION 487 20 HighThroughputScreeningTechniquesinProteinPurification 489 KarolM.LackiandEggertBrekkan INDEX 507 PREFACE TO THE THIRD EDITION Most will agree that the major achievement in bioscience leaner implementation as well as better control. Expression since 1998, when the second edition of this book was pub- ofmonoclonalantibodiesinmammaliancellsisatthemulti- lished, is sequencing of the human genome. Rather than gram per liter level, with cell densities of more than twenty diminishing interest in proteins, this has led to a revival millionpermilliliter,specificproductivityover20picograms in protein exploration and an intensive search for better per cell per day, in bioreactors with capacities up to 20,000 understanding of molecular processes in health and disease. liters. This several-hundred-fold increase in productivity Duringthistime,industrialexploitationofproteinsinhealth- has changed the pressures on downstream purification, care has hardly declined. The application of monoclonal resultinginthedevelopmentofveryhighcapacitychromato- antibodies targeted against rheumatoid arthritis and cancer graphymediaforproductcaptureandhighlyselectivemedia has been booming, many second- and third-generation bio- (frequently “multimodal”) for polishing. Downstream puri- pharmaceuticals have been approved, and modern technol- fication of biopharmaceuticals uses platform modules for ogies for vaccine production based on protein engineering assuring virus safety and for removal of host cell proteins, andcellculturearebeingdevelopedonawidefront. aggregates and critical contaminants. Regulatory agencies There are approximately 21,000 protein-encoding genes, are encouraging greater understanding and control of pro- andthehumanproteomeismuchlargerthanthis.Although duction processes, a quality by design (QbD) doctrine, and mapping the genome revealed what was in the box, the the use of modern risk management techniques and exper- jigsaw puzzle is far from complete. Several major research imental design—all of which is impacting the development projects exemplify the revitalized interest in proteins. One ofpurificationmethods. isthe Protein Atlas initiative (www.protematlas.org), aimed Comparedtothesecondeditionofthisbook,fourchapters at providing a comprehensive database of high resolu- havebeendeleted (Chromatofocusing,AffinityPartitioning, tion microscopic images identifying proteins in normal and Immunoelectrophoresis, and Large-Scale Electrophoresis). cancer tissues. Others involve an ever-widening range of Three chapters have been totally rewritten by new authors: refinedtoolsexploitingproteinprofilingmicroarrays,surface Chapter 5 (High Resolution Reversed-Phase Liquid Chro- plasmonresonance,massspectrometry,ELISA,quantitative matography of Proteins), Chapter 15 (Electrophoresis in 2Delectrophoresis,andsoon.Manytechnologiesareaimed Gels), Chapter 16 (Conventional Isoelectric Focusing in at parallel processing of thousands of targets, and this is Gel Slabs and Capillaries and Immobilized pH Gradients). profoundly changing the way structural biology projects Six new chapters have been added: Chapter 10 (Affinity are managed. Streamlined, miniaturized, automated high Ligands from Chemical Combinatorial Libraries), Chapter throughput (HTP) protocols are becoming the standard, but 11 (Affinity Ligands from Biological Combinatorial there is still a fundamental need for protein expression and Libraries), Chapter 12 (Membrane Separations), Chapter 13 purification, not least for X-ray structural studies. Many (RefoldingofInclusionBodyProteinsfromE.coli),Chapter “proteomic” projects exploit high throughput purification of 14 (Purification of PEGylated Proteins), and Chapter 20 taggedproteinsorantibodies. (High Throughput Screening Techniques in Protein Puri- Ontheindustrialside,particularinhealthcare,proteinpro- fication). These new chapters have been written by leading ductionisrapidlymaturing.Platformtechnologiesarebeing experts in their respective fields. All other chapters have applied both upstream and downstream, allowing fasterand been thoroughly revised and updated regarding recent vii viii PREFACE TO THE THIRD EDITION applications. A new section on the history of protein chro- all contributing authors and to Ms Anita Lekhwani matography has been added to Chapter 2 (Introduction to and her staff at John Wiley & Sons, Inc., Hoboken, New Chromatography). Jersey, for their patience and never-failing support of this It is my hope that the third edition will receive the same project. overwhelmingly positive response as the first and second editions, and I would like to express my appreciation to JAN-CHRISTERJANSON PREFACE TO THE SECOND EDITION Since1989,whenthefirsteditionofthisbookwaslaunched, As long as scientists have been engaged in the isolation thedevelopmentofbioscienceshasmeantarevivalofprotein and purification of proteins from crude extracts, there has chemistry in the wake of the molecular biology revolution beenademandformediawithhigheradsorptiveselectivities. and the HUGO project. The total genome of baker’s yeast Theextremelyhighvariabilityinproteinsurfacestructureas is now sequenced, that of E. coli is not far behind, and well as their wide range of functional stabilities, makes it within a not too distant future the feat of the total mapping necessaryforeveryproteinchemisttohaveastockofseveral of the human genome,which at the beginning seemed ficti- different separation media, ion exchangers, hydrophobic tious, is now within reach. This means that the attention of interaction media, and a variety of general affinity media. the world’s bioscientific community will again, as in the Literature survey data presented in some of the chapters of 1960sandmostofthe1970s,focusonthestructureandfunc- thisbookrevealthatonaveragesomewherebetweenthreeand tionoftheproteins.ThePROTEOMEerahasthusbegun,and fourstepsarerequiredtopurifyaproteintohomogeneity.The withitfollowstheneedofmoreefficientandmoreselective hope forone-step purifications raised by the introduction of toolsfortheseparation,isolation,andpurificationofthegene immobilized monoclonal antibodies has not yet been ful- products,theproteins. filled. However, there is a renewed opportunity at hand to The development of new chromatographic separation increase the selectivity of immobilized ligands in affinity media since 1989 has mainly been focused toward improve- chromatography and thus decrease the number of steps in mentsdemandedprimarilybyprocessdevelopmentengineers the purification process. This opportunity has been raised inthebiopharmaceuticalindustry.Thishasresultedinmedia by the recent rapid development in the design of a large withhigherefficiencies,leadingtoshortercycletimes,primar- variety of chemical and biological combinatorial libraries ily based on suspension polymerized styrene-divinylbenzene and high-speed screening technologies. It is easy to predict polymers with optimized internal pore size distributions, that over the next few years there will be an unprecedented some allowing partial convective flow through the particles. number of new highly selective ligands, monospecific as Thistrendhasreceiveditsultimatesolutionintotallyperfusive well as group specific, introduced for the synthesis of new systems basedonstackedmembranes, orcontinuous“mono- proteinseparationmedia. lithic” columns made of cross-linked polymers, derivatized Comparedtothefirsteditionofthisbook,thereexistsone withvariouskindsofproteinadsorptivegroups.Newcompo- additionalchapter(Chapter18)onlarge-scaleelectrophoretic site media have been introduced primarily to increase the processes. Three chapters (Chapters 15, 16, and 17) have industrial applicability of size exclusion chromatography of been totally rewritten. Chapters 15 and 16 by new authors. proteinsbutalsotoincreasebindingcapacityin,forexample, Most other chapters have been thoroughly revised, and all ion exchange chromatography. The concept of “solid diffu- havebeenupdatedregardingrecentapplications. sion” in highly ionic group substituted composite media is It is our hope that this newedition will receive the same stillawaitingitsphysicochemicalexplanation. overwhelmingly positive response as the first edition, and Thedemandforsystemsallowingdirectcaptureoftarget we would like to express our appreciation to Dr. Edmund proteins directly from whole cultures or cell homogenates, H. Immergut and the staff of VCH Publishers, now John resulting in fewer process steps and concomitantly higher Wiley&Sons,Inc.,fortheirpatienceandnever-failingsup- yields, has led to a revival of the fluidized bed concept. portofthisproject. However, now optimized with regard to the design of both JAN-CHRISTERJANSON mediaandcolumnsbytheintroductionofthemoreefficient LARSRYDE´N onecycletechniquecalledexpandedbedadsorption. ix PREFACE TO THE FIRST EDITION Over the last two decades the scientific community has Starting with this general concept, we have aimed at witnessedanunprecedentedexpansionwithinthebiosciences providing students, teachers and research workers in bio- andbiotechnology.Thisexpansionhasbeentoalargeextent medicine, bioscience and biotechnology with a concise and drivenbyadvancesinseveralkeyareas,mostnotablyrecom- practical treatise covering, in a single volume, all important binant DNA technology, hybridoma and cell culture tech- chromatographicandelectrophoretictechniquesusedinpre- niquesand,finally,inbiochemicalseparationmethods.This parativeandanalyticalproteinchemistry.Thebookcontains book is a description of the current status of one of these a general introductory chapter on protein preparative work, areas:moderntechniquesforproteinpurificationandanalysis. Chapter1,wherethekeyconceptsareintroduced.Similarly,a The research on which the progress in separation generalintroductiontochromatographyisgiveninChapter2 techniques is based has been conducted both in university and an introduction to analytical electrophoresis in Chapter departments, devoted to basic research, and in industrial 12. The major chromatographic and electrophoretic techni- laboratorieswhosemainconcernisthedevelopmentofnew quesarepresentedinindividualchapters,includingonechap- equipment and tools. In many cases the two communities ter on affinity partitioning in aqueous polymer two-phase have cooperated to their mutual benefit. In fact, a great systems. number of the products now available for the separation Nosinglepersoncantodaybeevenclosetoacquiringthe andpurificationofproteins, suchaschromatographicmedia amountofexperiencenecessarytodescribewithconfidence with a wide range of selectivities and efficiencies, as well the wealth of techniques and methods which makes up the as equipment for electrophoretic separation and analysis, arsenalforproteinseparations.Wehavethuschosentopro- were originally developed in a universitysetting. This book duce a multi-author volume recruiting expertise from the isalsotheresultofajointeffortbetweenuniversityresearch- entire field. All chapters have, however, been thoroughly ers,inparticularatUppsalaUniversity,andtheresearchstaff worked through by the editors to achieve a reasonable uni- ofacompany,PharmaciaLKBBiotechnology.Althoughitis formity of style and organization. Each chapter deals first thusaproductofthisconditionofmutualbenefit,theambi- withthetheoryandunderlyingprinciplesofeachseparation tion has not been to give a selective description of methods technique,followed bya section on methodology, and ends or materials from a single commerical source, but rather to with a number of representative application examples giveanunbiasedaccountofallkeytechniquesinthefield. describedindetail. Todayitistoagreatextentpossibletobasetheseparation Thepreparationofthisbookhasbeenamatterofseveral ofproteinsonknowledgeoftheirmolecularproperties,struc- years. We would like to thank the authors for their turalaswellasfunctional.Suggestionsonhowtosolveasep- cooperation, from the first planning stage to the last phase arationproblemcanbestbemadeifdataonproteinstructure ofupdatingandaddition.Wewouldalsoliketothankouredi- andfunction, including particular structuraldetails,is avail- tors at VCH Publishers in New York, in particular Dr. able.Conversely,resultsfromtheapplicationofaparticular Edmund H. Immergut who took the first initiative and who separation method can often be interpreted in terms of mol- followed the project up to its realization. The management ecularpropertiesoftheproteinunderstudy.Throughoutthe and staff of Pharmacia LKB Biotechnology are thanked for textofthisbook,separationresultsarerelatedtoproteinprop- their cooperation and support which allowed the selling erties,ofteninadetailedmanner.Wearethefirstgeneration price to be considerably reduced. Manystaff members have tobeonthevergeofrationalproteinmanagement. made invaluable contributions to the final result, which are xi xii PREFACE TO THE FIRST EDITION gratefully acknowledged. We also thank Elizabeth Hill and oftheotherauthorsofthisbook,springfromthetreeplanted Ursula Snow for their contributions in the early phase of longagobyTheSvedbergandArneTiselius,andlaterkept the project; Inger Galve´r, Gull-Maj Hede´n, Inga Johansson alive by Jerker Porath and Stellan Hjerte´n and many of and Madeleine de Sharengrad for secretarial work; Bengt their colleagues and pupils through fifty years of separation Westerlund for handling the computer programmes for the science at Uppsala University. We offer this book as the chemicalstructures;UnoSkattandLilianForsbergforprodu- latestfruitofthistree,hopefullytobeenjoyedbymany. cinganumberoftheillustrations;andDavidEakerandJohn Brewer for keeping our freedom with the English language withinlimits. Finally,wewouldliketoaddthatwearewell JAN-CHRISTERJANSON awarethatmuchofourownefforts,occasionalachievements LARSRYDE´N andsometimeshardwonexperience,aswellasthatofseveral Uppsala,Sweden,June21,1989
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