生物分离工程 课件-双语 Chapter 7-Chromotography-2-Hydrophobic interaction

Chapter8-2Hydrophobic

Interaction

Chromatography（HIC）1Introduction2下村修钱永健马丁They

all

faced

the

challengesabout

purifying

GFP

from

different

sources.GFPPurificationProcessExpressionCell

cultureCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation3将GFP基因插入大肠杆菌表达系统超声波破碎过滤澄清硫酸铵沉淀，透析离子交换色谱（如Sepharose-DEAE）微滤灭菌，装罐细胞培养、诱导表达、蛋白扩增ExpressionCell

cultureCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation4平衡、结合、洗涤：pH值：pH=7.4（大于PI）离子强度：10mMNaCl（低盐）洗脱：pH值：pH=5.5离子强度：500

mMNaCl色谱柱：类型：阴离子交换柱（Sepharose-DEAE）柱体积：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：pH值：pH=3（极端pH，兼顾树脂）离子强度：1

MNaCl（高盐）最后换成纯水清洗GFPPurificationProcessChromatographyCurve平衡—上样—洗涤—洗脱—再生GFPGradient:

0%to50%elutionbufferover10mL(10CV)DiscussionWhatifthereareother

proteinsthathavesimilarchargetoGFPinIEC-elutedsolution?GFPOtherproteinsChapter8-2Hydrophobic

Interaction

Chromatography（HIC）7OverviewFactorsinfluencingseparationeffectApplicationOutline8Overview—Principle9Separationisachievedviathereversible

andhydrophobicinteractionsbetweenthe

hydrophobic

region

of

proteins

andthestationaryphase.Proteinsareseparatedbasedonthehydrophobicbindingstrengthwith

the

resin.

Hydrophobicbinding

strengthispositively

relatedtothe

surfacehydrophobicityof

proteins

and

saltconcentration.10Overview—PrincipleRed:

Hydrophilic

amino

acidsYellow:

Hydrophobic

amino

acids

ReversibleinteractionSalt

can

disrupthydrationshellof

proteins

and

enhancethe

hydrophobiceffect.Moleculesareloaded

at

ahighsaltconcentrationsolution,and

eluted

byreducingthesaltconcentration

to

weaken

thehydrophobicinteractions11Overview—PrincipleHIC

separatesbiomoleculesbasedonthedifferenceinhydrophobicinteractions

betweenproteinsandthehydrophobicadsorbentusedasthestationaryphase.Accordingto“saltingout”,inhighsaltconcentrationsolutions,thehydrationlayerofproteinisdisrupted,thus

exposingthehydrophobicpartsandincreasinghydrophobicinteractions.Moleculesareboundtothecolumnusingahighsaltconcentrationsolution,and

eluted

byreducingthesaltconcentration

to

weaken

thehydrophobicinteractions12Overview—PrincipleHIC

isa

supplementtootherproteinseparationmethods

basedonproteincharge,size,andligand-receptor

recognition,andiswidelyusedinproteinpurification.It

isanidealdownstreamstepforsamplesafterammoniumsulfateprecipitationorionexchangechromatography,

which

containshighsalt

concentrationstobedirectlyloadedontotheHIC.Duringtheseparation,thesampleispurifiedandelutedinsmallvolumes,makingitpossibletodirectlyloadtheconcentratedsampleontogelfiltrationchromatographyorother

chromatographyafterbufferexchange.13Overview—CharacteristicsHIC

resinconsistsoftwoparts:carrier(matrix)

and

functionalgroups

(ligand).14Overview—FormationHydrophobicresin-polystyreneresin聚苯乙烯树脂Hydrophilicpolymer纤维素(Cellulose)球状纤维素(Sephacel)葡聚糖(Sephadex)SephadexG-25和G-50琼脂糖(Sepharose)SepharoseCL-6B1.

Carrier(matrix)15Overview—Formation琼脂糖类凝胶仍是应用最广泛的疏水介质ThehydrophobicligandsusedinHICaremainlyalkyl

(烷基)andaromatic

(芳香基)groups.ThealkylgroupsareusuallyshorterthanC8,whilethearomaticgroupsareoftenphenyl

(苯基)groups.

R:

Hydrophobicligands，M:

Carrier2.

Functionalgroups(ligand)16Overview—Formation（A）丁基Butyl（B）辛基Octyl（C）苯基Phenyl（D）新戊基Pentyl

2.

Functionalgroups(ligand)Commonly-used17Overview—FormationSome

commercialHIC

resins.1.

ButylSepharose4FastFlow正丁烷基，疏水性最弱，适合含脂族配体的生物分子。2.OctylSepharose4FastFlow正辛烷基，疏水性中等，适合各种蛋白的分离纯化。⒊PhenylSepharose6FastFlow苯基Phenyl，疏水性最强，载量高，适合含芳香族配体的生物分子的预处理，2.

Functionalgroups(ligand)18Overview—FormationDiscussion19ExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECFormulation微滤OtherproteinsDiscussion20ExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECHICFormulation将GFP基因插入大肠杆菌表达系统超声波破碎过滤澄清硫酸铵沉淀，透析DEAE,

pH=7.4,低盐上样，高盐洗脱微滤疏水作用色谱浓缩，灭菌装瓶色谱条件和过程如何选择？1.

Stationaryphase

(HIC

Resin).2.Mobile

phase.3.Chromatographicconditions.Factorsinfluencingseparationeffect211.HIC

ResinFactors:

①carrier,

②ligand

type,

③ligand

density.Carriers

usedinHICresinaremainlypolysaccharides(suchasagarose琼脂糖)andsyntheticpolymers(suchaspolyethylene聚乙烯andpolyvinylalcohol聚乙烯醇).Factorsinfluencingseparationeffect22大分子——无孔小粒径填料小分子——多孔填料1.HIC

ResinFactors:

①carrier,

②ligand

type,

③ligand

density.Carriers

usedinHICresinaremainlypolysaccharides(suchasagarose琼脂糖)andsyntheticpolymers(suchaspolyethylene聚乙烯andpolyvinylalcohol聚乙烯醇).Ligand

type

includes

alkyl烷基andaromatic芳香基groups.Generally,thebindingcapacity(hydrophobicity)increaseswithincreasingalkylchainlength.Factorsinfluencingseparationeffect2324这四种不同的配基条件是:苯基(高取代)，苯基(低取代)，辛基和丁基。Ligand

Type25Ligand

density

(substitutiondegree):

It

affectsthebindingcapacity.Increasing

ligand

densityresultinmorebindingsitesforproteins,therebyincreasingthebindingcapacity.However,whenitreachesacertainvalue,thesterichindrancelimit

the

further

increase

of

bindingcapacity.And

multiple

ligandsboundtoeachprotein,resultinginoverlystrongbindingthatisdifficulttoelute.FactorsinfluencingseparationeffectLow

ligand

densityModerate

ligand

densityHigh

ligand

densityLow

binding

capacityHard

to

eluteMaximumBindingand

easyto

eluate26Factors:1)Ion

typeandionic

strength.2)pH.3)Additives

(添加剂).2.Mobile

phaseFactorsinfluencingseparationeffect27Sodium/ammoniumsulfatecanpromoteligand-protein

interactioninHIC.

So,

Themostcommonlyusedsalts:

Na2SO4

and

(NH4)2SO4AnionssuchasClO4-andI-withsmallionicradiiandlowchargedensitycanweakenthehydrophobicinteraction,

whichcan

be

usedduringelution.

Factorsinfluencingseparationeffect2.Mobile

phase

—

①Iontypeandionicstrength282.流动相组成

1）离子种类及强度Ion

type

matters!29Load

thesampleatasaltconcentrationslightlybelowthesalting-outpoint.在略低于盐析点的盐浓度上样吸附Graduallyreducetheionicstrengthofthemobilephaseduringelution.洗脱时逐渐降低流动相的离子强度Factorsinfluencingseparationeffect2.Mobile

phase

—

①Iontypeandionicstrength3031FactorsinfluencingseparationeffectIonic

strength

matters!32FactorsinfluencingseparationeffectThechoiceofpHmustbecompatiblewithproteinstabilityandactivity.ThepHcanaffectthesurfacechargeof

theproteins.Generally,apHvalueclosetotheprotein'sisoelectricpoint(pI)ischosentoreduceelectrostaticinteractionsbetweenproteins,whichisbeneficialforimprovingseparationperformance.Factorsinfluencingseparationeffect2.Mobile

phase

—

②pH33Additivescanbeusedtoimproveselectivity/resolution.Theyaretypicallyusedtopromoteproteinsolubilizationandalterproteinconformationtofacilitateelutionwhensampleandhydrophobicinteractionchromatographypackingmaterialsaretightlybound.Factorsinfluencingseparationeffect2.Mobile

phase

—

③Additives34Factors:

Temperature,

Flow

rate,

etc.3.

ChromatographicconditionsFactorsinfluencingseparationeffectHydrophobicbindingforceincreaseswithincreasingtemperature.35Generally,thehighertheflowrate,theworsetheseparationefficiency.3.

ChromatographicconditionsFactorsinfluencingseparationeffect36DiscussionThemolecularweightofGFPis27kDaanditsisoelectricpointis5.76,anditremainsstableinpH

from

5.5

to

8.

Please①updatetheIEC&HIC-basedGFPpurificationprocess.IncludetheselectionofHICresin,loadingandelutionconditions,andotherspecificparameters.②Drawthepotentialchromatographycurve.（设计纯化工艺，写清楚层析条件，画出层析曲线）37IECHICDiscussionExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECHICFormulation平衡、结合、洗涤：pH值：pH=7.4（大于PI）离子强度：10mMNaCl（低盐）洗脱：pH值：pH=5.8离子强度：10mMNaCl,

1

M

(NH4)2SO4

色谱柱：类型：阴离子交换柱（Sepharose-DEAE）柱体积：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：pH值：pH=3（极端pH，兼顾树脂）离子强度：1

MNaCl（高盐）最后换成纯水清洗Discussion39ExpressionCell

disruptionClarificationPrecipitation,

DialysisMicro-filtrationIECHICFormulation平衡、结合、洗涤：pH值：pH=6.0（稳定）盐：10mMNaCl,

1

M

(NH4)2SO4

（高盐）体积：10倍柱体积洗脱：pH值：pH=7.4盐：10mMNaCl（低盐）梯度：0-50%洗脱液（10倍柱体积）色谱柱：类型：苯基低取代（Sepharose-Phenyl）柱体积：2

mL

(1~100

mL)流速：2

mL/min（1~10

mL/min）再生：低盐/纯水清洗，加入表面活性剂Discussion40GFP

Purification-141GFP

Purification-242GFP

Purification-343GFP

Purification-444GFP

Purification-545GFP

Purification-646GFP

Purification47DiscussionExpressionCell

disruptionClarificationDialysis

&

Mi