Protein Purification: Principles, High Resolution Methods, and Applications (Methods of Biochemical Analysis)

522 Pages·2011·17.73 MB·English

by  Jan-Christer Janson

PROTEIN PURIFICATION
PROTEIN PURIFICATION
Principles, High Resolution Methods, and Applications
Third Edition
Editedby
JAN-CHRISTER JANSON
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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