蛋白质纯化的原理和方法

1、蛋白质纯化的原理和方法（Protein Purification Principles and Methods）ProteinsComplex, polymeric, asymmetric and sensitive moleculesContain covalent bound prosthetic groups and non-covalent bound cofactors Many non-covalent bounds e.g. Hydrogen-Bounds, Dipol-Interactions and Hydrophobic-Interactions“Weak” interac

2、tions are important for structure and function (activity) of the protein In most cases the purification must be gentle!Before the purificationCultivation of bacteriaCell disruption: Periplasmic and cytoplasmic proteins are releasedCentrifugation leads to a soluble fraction(supernatant) which contain

3、s all soluble periplasmic and cytoplasmic proteins and a membrane fraction from which membrane bound proteins can be solubilised with detergents (e.g. Triton X-100)The soluble or membrane fraction are the start point of the further purification by chromatographyCell disruption:French PressLysozymeUl

4、trasonicFrench PressMembrane ProteinsPeripheral membrane proteins: in most cases soluble in buffers with high or low ionic strength or high pHIntegral membrane proteins: they contain trans membrane helices and must be solubilised to conserve conformation and function of the proteinSolubilisationof i

5、ntegral membrane proteinsSolubilisationof proteins is done with a detergents concentration above the CMC to ensure the incorporation of membrane lipid into detergent micelles.CMC = critical micelle concentrationdepends on temperature, ionic strength and pH of the buffer and concentration of uncharge

6、d substances like urea or alcoholSome detergentsIonic detergents:Sodium-Dodecylsulfate:denatures Proteins (SDS-PAGE)Na-Deoxycholate: preticipatesby pH<6.8preticipatewith Ca2+or Mg2+In General: No ionic Detergents in purifications with depend on the charge of the proteins. Non-ionic detergents:Tri

7、ton X-100 Phenyl groups strong Absorbance at 280 nmTween 20 like Triton X-100 not dialyzableZwitter-ionic detergentsChaps dialyzableWhich proteins are purified?Metabolic pathwaysEnergy productionAim: biochemical characterisation (Reactivity, subunit composition, organic and inorganic cofactors, 3D s

8、tructure)and why?Purification strategiesProtein stabilisation:Integral membrane Proteins: SolubilisationPurification at 4°C: reduces protease activityAddition of protease inhibitors: commonly used are EDTA and PMSF (toxic)Quickly load on first column after cell disruption and ultra centrifugeMa

9、in impurities are removed first, lesser in the second or thirdstepMax 5% impurities are acceptableIn general:Max 4 purification stepsNo steps with purification factor < 5No steps with < 30% yieldNo steps which last longer than one day and one night ChromatographySeparation material in columns,

10、 streamed by buffer liquid chromatographyHPLC: high pressure/performance liquid chromatographyFPLC: fast protein liquid chromatographyFPLC unitSeparation principlesSize:size exclusion chromatography (= gel filtration, = gel permeation chromatography) Chargeanion or cation exchange chromatography; ch

11、romatofocusingHydrophobicityhydrophobic interaction chromatography (HIC)Affinityaffinity chromatographySolubilityammonium sulfate precipitation (non chromatographic, rel. imprecise)Size exclusion chromatographyDifferent pore sizes, depending on the size of the proteins Separation is based in diffusi

12、on slow flow rate Pressure sensitive materialsExample for a separation by gel filtrationNoteworthy about chromatographyGel filtration:limited sample volume: 2-3mllimited flow rate gel filtraion takes timedelutionof the sample by a factor of about 3low purificatopn factor: 3-6needs column buffer with

13、 high ionic strength: min 0.1 MIon exchage chromatography:To remember:Protein binding depents on electrostatic interactions with the columnmaterial.Strength of binding depents on pH and ionic strength of the buffer, the pI of the protein and the charge density on the column.In general:Technical easi

14、er than gel filtration: columns could be packed at the FPLC.Sample volume can be multiple times the column volume.Higher flowrates.Purification factor: 3-15Sample gets concentrated.Dont use charged detergents!Ion exchage chromatographyBinding behaviour of proteinspI= isoelectricpoint of the protein

15、the pH value at which the posses no net charge The pI determines the charge of the protein at a given pHpH > pI negative net charge pH < pIpositive net chargepIand protein separation on ion exchange columnsBut.pIis not alone responsible for the binding behaviour of proteinsBinding is sometimes

16、 influenced by local charges and not by thenet charge of the proteinExample for a separation by ion exchange chromatographyMaterialsCarrier materialsPoly sugarsSepharose (Agarose), Sephadex (Dextran), Sephacel (Cellulose) Rare sugars can hardly be utilised by bacteria.Low flow ratesMaterial changes

17、its Volume depending on the ionic strangth Material settles over timeBeadsPolystyrol/Divinylbenzen beads with charge carrier addedRelative high flow ratesGood pressurestability, no compressionHard charges could stress the proteinBeads and tentacleCharges are linked with flexiblespacers to polymeric beads. This