Inclusion Bodies: Methods and Protocols

275 Pages·2023·7.959 MB·English

by  Julian Kopp, Oliver Spadiut

Methods in
Molecular Biology 2617
Julian Kopp
Oliver Spadiut Editors
Inclusion
Bodies
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
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Inclusion Bodies
Methods and Protocols
Edited by
Julian Kopp and Oliver Spadiut
Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Vienna, Austria
Editors
JulianKopp OliverSpadiut
InstituteofChemical,Environmental InstituteofChemical,Environmental
andBioscienceEngineering andBioscienceEngineering
TUWien TUWien
Vienna,Austria Vienna,Austria
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-2929-1 ISBN978-1-0716-2930-7 (eBook)
https://doi.org/10.1007/978-1-0716-2930-7
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Preface
Escherichia coli is a commonly used host organism in academia and industry, due its
sequenced genome, well-characterized physiology, and rapid doubling rates in cheap and
defined media. Thus around 25% of all recombinant biopharmaceuticals are produced in
E. coli to date. In many E. coli processes, recombinant proteins are expressed as inclusion
bodies(IBs).Theseintracellularproteinaggregatesareformedeitherduetorapidrecombi-
nant protein synthesis and insufficient availability of chaperones or due to product-specific
structures,whichcannotbeprovidedbyE.coli,likeproteinglycosylation.
In the past, IBs were considered to be non-functional waste products, and various
strategiesweredevelopedtoreducetheir formation.However,inthepast20years,numer-
ous studies have demonstrated that proteins enclosed in IBs can be biologically active,
makingIBformationanattractivestrategytoproducelargeamountsofbioactiverecombi-
nantprotein.
BesidesthedirectuseofcatalyticallyactiveIBs,thefast-emergingmarketofbiosimilars
inthebiopharmaceuticalindustryhasahighdemandforrecombinanttherapeuticproteins.
To ensure steady medical supply and enable economic manufacturing, it is of the utmost
importance to enhance yield, accelerate process development time, and lower production
costs.Consideringtheseaspects,theproductionofrecombinantbiopharmaceuticalsinthe
form of IBs instead of soluble protein presents a highly attractive option with many
advantages, such as high product yield (more than 20 g IBs/L cultivation broth), short
production times, high product purity in IBs (up to 95%), high mechanical and thermal
stability,andresistancetoproteases.
However, in order to obtain refolded, active biopharmaceutical protein, a complex
multi-step process is usually required. First, IBs are produced in E. coli, typically in
fed-batch cultivations. Then, the broth is harvested and the cells are mechanically lysed,
e.g., by sonication or high-pressure homogenization. Consecutive IB processing steps
comprise IB washing, solubilization with strong chaotropic denaturants, and refolding of
the product in its native conformation. The latter step is usually performed by batch
dilution, dialysis, or via on-column refolding. This multi-step process comes with several
critical challenges, like the need of harsh chaotropic agents in high concentrations, long
developmenttimes,highvariability,unpredictability,andverylargebuffer volumes.
This book comprises a total of 19 contributions dealing with different aspects of IB
processing – from cloning to purification of refolded product – and presenting novel
methodsinIBtechnology.
TheintroductorychaptersummarizesthecurrentstateoftheartinIBprocessing.
In Chaps. 2, 3, 4, and 5, upstream processing is addressed highlighting thermo-
inducible systemsfor IB production,automated roboticplatforms for high-throughputIB
expression, production of bioactive IBs, and novel bi-directional promotor systems for IB
production.InChaps.6and7,theprocessingmodeoffed-batchcultivationandcontinuous
cultivationforIBproductionisaddressedhighlightingonlineflowcytometryasamonitor-
ingandcontroltoolforcultivationprocesses.
v
vi Preface
Chapters8,9,10,11,12,13,14,15,and16describeIBprocessingstepsincludingcell
disruption techniques, diverse IB solubilization and refolding techniques, as well as the
monitoringofsecondaryproteinstructuresinIBs.
Thelastfewchapters,Chaps.17,18,and19,describedifferentIBsuccessstories.
The protocols given in this book shall describe and facilitate different IB processing
steps.
Vienna,Austria JulianKopp
OliverSpadiut
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 InclusionBodies:StatusQuoandPerspectives....... ........ ....... ........ 1
JulianKoppandOliverSpadiut
PART I UPSTREAM PROCESSING
2 ThermoinducibleE.coliforRecombinantProteinProduction
inInclusionBodies......... ........ ....... ....... ........ ....... ........ 17
NormaA.Valdez-CruzandMauricioA.Trujillo-Rolda´n
3 High-ThroughputExpressionofInclusionBodies
onanAutomatedPlatform.......... ....... ....... ........ ....... ...... .. 31
AnninaKemmer,LindaCai,M.NicolasCruzBournazou,
andPeterNeubauer
4 Design,Production,andCharacterizationofCatalytically
ActiveInclusionBodies..... ........ ....... ....... ........ ....... ........ 49
GizemO¨lc¸u¨cu¨,Karl-ErichJaeger,andUlrichKrauss
5 BidirectionalPromoterLibrariesEnabletheBalanced
Co-expressionofTwoTargetGenesinE.coli ....... ........ ....... ........ 75
JuliaJodlbauer,LukasRieder,AntonGlieder,andBirgitWiltschi
6 InclusionBodyProductioninFed-BatchandContinuous
Cultivation......... ....... ...... .. ...... ...... .. ........ ....... ........ 87
StefanKittler
7 AvoidingtheAll-or-NoneResponseinGeneExpression
DuringE.coliContinuousCultivationBasedontheOn-Line
MonitoringofCellPhenotypicSwitchingDynamics ......... ....... ........ 103
FrankDelvigne,LucasHenrion,VincentVandenbroucke,
andJuanAndresMartinez
8 OptimizationofInclusionBodyFormationandPurification
inMulti-wellPlates......... ........ ....... ....... ........ ....... ........ 121
SarahKamel,JuliaSchollmeyer,AnkeKurreck,andPeterNeubauer
PART II DOWNSTREAM PROCESSING
9 HighPressureHomogenizationforInclusionBodyIsolation ........ ........ 141
JulianEbner,ViktorSedlmayr,andRobertKlausser
10 DevelopmentofSolubilizationandRefoldingBuffers........ ....... ........ 155
MohammadRoufarshbafandVajiheAkbari
11 UnitOperation-SpanningInvestigationoftheRedoxSystem ........ ........ 165
JulianEbner,DianaHumer,andViktorSedlmayr
vii
viii Contents
12 UsingHighPressureandAlkalinepHforRefolding ......... ....... ........ 177
LigiaMorgantiandRosaMariaChura-Chambi
13 IntensificationofInclusionBodyProcessingvia
Temperature-BasedRefolding....... ....... ....... ........ ....... ........ 189
RuShenWong,NurulNadiaMohamadAlias,
EugeneBoonBengOng,andMervynW.O.Liew
14 RefoldingofProteinsExpressedasInclusionBodiesinE.coli........ ........ 201
RashmiSharma,AnupaAnupa,andAnuragS.Rathore
15 InfraredSpectroscopyforStructureAnalysisofProtein
InclusionBodies.... ....... ........ ....... .... ... ...... .. ....... ........ 209
AndreasSchwaighoferandBernhardLendl
PART III SUCCESS STORIES
16 ThePurificationofHemePeroxidasesfromEscherichiacoli
InclusionBodies:ASuccessStoryShownbytheExample
ofHorseradishPeroxidase .......... ....... ....... ........ ....... ........ 227
DianaHumerandJulianEbner
17 Productionofα-SynucleinFibrillar-SpecificscFv
fromInclusionBodies...... ........ ....... ....... ........ ....... .. ..... . 239
VijayGupta,IssamHmila,NishantN.Vaikath,
IndulekhaP.Sudhakaran,andOmarM.A.El-Agnaf
18 MethodforInclusionBodiesProductionviaE.coli
HostSystem:rGCSFasModelBiotherapeuticProtein ....... ....... ........ 249
AbhilashaK.Rani,VenkateswaraR.Naira,andAnuragS.Rathore
19 AntimicrobialApplicationsofInclusionBodies...... ........ ....... ........ 257
RamonRoca-Pinilla,AnnaArı´s,andElenaGarcia-Fruit(cid:1)os
Index .......... ........ ....... ........ ....... ....... ........ ....... ........ 271
Contributors
VAJIHEAKBARI • DepartmentofPharmaceuticalBiotechnology,FacultyofPharmacy,Isfahan
UniversityofMedicalSciences,Isfahan,Iran
NURUL NADIAMOHAMADALIAS • InstituteforResearchinMolecularMedicine
(INFORMM),UniversitiSainsMalaysia,Penang,Malaysia
ANUPAANUPA • SchoolofInterdisciplinaryResearch,IndianInstituteofTechnologyDelhi,
NewDelhi,India
ANNAARI´S • DepartmentofRuminantProduction,InstitutdeRecercaiTecnologia
Agroaliment`aries(IRTA),CaldesdeMontbui,Spain
LINDACAI • TechnischeUniversita€tBerlin,InstituteofBiotechnology,ChairofBioprocess
Engineering,Berlin,Germany
ROSAMARIACHURA-CHAMBI • CentrodeBiotecnologia,InstitutodePesquisasEnerge´ticas
eNucleares,IPEN-CNEN/SP,Sa˜oPaulo,SP,Brazil
M.NICOLAS CRUZBOURNAZOU • TechnischeUniversita€tBerlin,InstituteofBiotechnology,
ChairofBioprocessEngineering,Berlin,Germany;DataHowAG,Du¨bendorf,Switzerland
FRANKDELVIGNE • TerraResearchandTeachingCentre,MicrobialProcessesand
Interactions(MiPI),GemblouxAgro-BioTech,UniversityofLie`ge,Gembloux,Belgium
JULIAN EBNER • IBDGroup,InstituteofChemical,EnvironmentalandBioscience
Engineering,TUWien,Vienna,Austria;TUWien,InstituteofChemical,Environmental
andBioscienceEngineering,ResearchAreaBiochemicalEngineering,Vienna,Austria
OMARM.A.EL-AGNAF • NeurologicalDisorderResearchCenter,QatarBiomedical
ResearchInstitute(QBRI),HamadBinKhalifaUniversity(HBKU),QatarFoundation,
Doha,Qatar
ELENAGARCIA-FRUITO´S • DepartmentofRuminantProduction,InstitutdeRecercai
TecnologiaAgroaliment`aries(IRTA),CaldesdeMontbui,Spain
ANTONGLIEDER • InstituteofMolecularBiotechnology,GrazUniversityofTechnology,Graz,
Austria
VIJAYGUPTA • NeurologicalDisorderResearchCenter,QatarBiomedicalResearchInstitute
(QBRI),HamadBinKhalifaUniversity(HBKU),QatarFoundation,Doha,Qatar
LUCASHENRION • TerraResearchandTeachingCentre,MicrobialProcessesandInteractions
(MiPI),GemblouxAgro-BioTech,UniversityofLie`ge,Gembloux,Belgium
ISSAMHMILA • NeurologicalDisorderResearchCenter,QatarBiomedicalResearchInstitute
(QBRI),HamadBinKhalifaUniversity(HBKU),QatarFoundation,Doha,Qatar
DIANA HUMER • IBDGroup,InstituteofChemical,EnvironmentalandBioscience,TU
Wien,Vienna,Austria;TUWien,InstituteofChemical,EnvironmentalandBioscience
Engineering,ResearchAreaBiochemicalEngineering,Vienna,Austria
KARL-ERICHJAEGER • InstituteofMolecularEnzymeTechnology,HeinrichHeineUniversity
Du¨sseldorf,ForschungszentrumJu¨lich,Wilhelm-Johnen-Straße,Ju¨lich,Germany;Institute
ofBio-andGeosciencesIBG-1:Biotechnology,ForschungszentrumJu¨lichGmbH,Wilhelm-
Johnen-Straße,Ju¨lich,Germany
JULIAJODLBAUER • acib-AustrianCentreofIndustrialBiotechnology,Graz,Austria
SARAHKAMEL • TechnischeUniversita€tBerlin,InstituteofBiotechnology,ChairofBioprocess
Engineering,Berlin,Germany
ix