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)Protei ns?Complex, polymeric, asymmetric and sen sitive molecules?Contain covale nt bound prosthetic groups and n (-covale nt bound cofactors ?Ma ny non-covale nt bounds e.g. Hydroge n-Bo un ds, Dipol-In teract ions and Hydrophobic- ln teract i

2、ons? “Weal” interactions are important for structure and function (activity) of the protein In most cases the purification must be gentle!Before the purificati on ?Cultivation of bacteria?Cell disrupti on: Periplasmic an dcytoplasmic prote ins are released?Centrifugation leads to a soluble fraction(

3、supernatant) which contains all soluble periplasmic and cytoplasmic prote ins and a membra ne fracti on from which membra ne bound prote in s can be solubilised with deterge nts (e.g. Trit on X-100)?The soluble or membrane fraction are the start point of the further purification by chromatographyCel

4、l disrupti on:French PressLysozymeUltras onicFrench PressMembra ne Protei ns/Peripheral membra ne prote ins: innost cases soluble in buffers with high or low ionic stre ngth or high pH?ln tegral membra ne protei ns: they contain trans membra ne helices and must be solubilised to con serve con format

5、i on and fun cti on of the proteinSolubilisati onof in tegral membra ne protei ns?Solubilisationof protei ns is done with a deterge nts concen trati on above the CMC to en sure the in corporati on of membra ne lipid in to deterge nt micelles.?CMC = critical micelle concen trati ondepends on temperat

6、ure, i onic stre ngth and pH of the buffer and concen trati on of un charged substa nces like urea or alcoholSome deterge nts?lonic deterge nts:Sodium-Dodecylsulfate:de natures Protei ns (SDS-PAGE)Na-Deoxycholate: preticipatesby pH6.8preticipatewith Ca2+or Mg2+In General: No ionic Detergents in puri

7、fications with depend on the charge of the prote ins. ?Noni onic deterge nts:Trit on X-100 Phe nyl groups strong Absorba nee at 280 nmTween 20 like Triton X-100 not dialyzable?Zwitter-i onic deterge ntsChaps dialyzableWhich prote ins are purified?Metabolic pathways?En ergy product ionAim: biochemica

8、l characterisati on (Reactivity, sub unit compositi on, orga nic and inorganic cofactors, 3D structure)a nd why?Purification strategies?Protein stabilisatio n:-ntegral membrane Proteins: Solubilisation-Purification at 4 C: reduces protease activitylAdditi on of protease in hibitors: com mon ly used

9、are EDTA and PMSF (toxic)-Quickly load on first column after cell disruption and ultra centrifuge?Main impurities are removed first, lesser in the second or thirdstep ?Max 5% impurities are acceptableIn general:Max 4 purification stepsNo steps with purification factor 5No steps with pI n egative net

10、 charge pH pIArtjcrd to on exchangersRange of&tabilityexchangersu5odccbuELIoOJZpositive net chargepland prote in separati on on ion excha nge colu mnsS 朮 cMty pH of mobile phaseBut.?plis not alone resp on sible for the binding behaviour of prote insBinding is sometimes in flue need by local charges

11、and not by the net charge of the proteingovrlinnl hfliontot 汕t rMluvl Lf htOFi1 t k缸 QM上*Mgf Mil washk1tgMly tisuhdi 忻uiLtt m 11 h&nij ibLuriil rrltiijikKiCMto畑 graawrt twginfin11xIXi2 C ”一警* =i二r ” TO-TO K2 GVuCckimncjExample for a separati on by ion excha nge chromatography* Sort anion exchangerDE

12、AE. DE-52jCHrCH2i+H(C2Hs24 Hard anion exchanger TMAE. McnoQ. SourceQ|ch厂ch尹叭c:比h-Soft cation exchanger Carboxymethyl|CH 厂 COO Hard cation exchangerSuffopropyt, MonoS. SourceS I 跆小厂沁，MaterialsCarrier materials?Poly sugars-Sepharose (Agarose), Sephadex (Dextra n), Sephacel (CelluloseRare sugars can ha

13、rdly be utilised by bacteria.-Low flow rates-Material changes it s Volume depending dxn strariigrth -Material settles over time?Beads-Polystyrol/Di viny Ibe nzen beads with charge carrier added-Relative high flow rates -Good pressurestability, no compressi on-Hard charges could stress the protein?Beads and ten tacle-Charges are lin ked with flexiblespacers to polymeric beads. This leadsto a soft binding of proteins despite of hard charges on the matrixR