蛋白质纯化的原理和方法（Protein Purification Principles and Methods）
Proteins
•Complex, polymeric, asymmetric and sensitive molecules
•Contain covalent bound prosthetic groups and non-covalent bound cofactors
•Many non-covalent bounds e.g. Hydrogen-Bounds, Dipol-Interactions and Hydrophobic-Interactions
•“Weak” interactions are important for structure and function (activity) of the protein
In most cases the purification must be gentle!
Before the purification…
•Cultivation of bacteria
•Cell disruption: Periplasmic an d cytoplasmic proteins are released
•Centrifugation leads to a soluble fraction(supernatant) which contains 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 chromatography
Cell disruption:French Press
Lysozyme
Ultrasonic
French Press
Membrane Proteins
•Pe ripheral membrane proteins: in most cases soluble in buffers with high or low ionic strength or high pH
•Integral membrane proteins: they contain trans membrane helices and must be solubilised to conserve conformation and function of the protein
Solubilisationof integral membrane proteins
•Solubilisationof proteins is done with a detergents concentration above the CMC to ensure the incorporation of membrane lipid into detergent micelles.
•CMC = critical micelle concentration
depends on temperature, ionic strength and pH of the buffer and concentration of uncharged substances like urea or alcohol
Some detergents
•Ionic detergents:
Sodium-Dodecylsulfate:denatures Proteins (SDS-PAGE)
Na-Deoxycholate: preticipatesby pH<6.8
preticipatewith Ca2+or Mg2+
In General: No ionic Detergents in purifications with depend on the charge of the proteins. •Non-ionic detergents:
Triton X-100 Phenyl groups strong Absorbance at 280 nm
Tween 20 like Triton X-100 not dialyzable
•Zwitter-ionic detergents
Chaps dialyzable
Which proteins are purified?
•Metabolic pathways
•Energy production
Aim: biochemical characterisation (Reactivity, subunit composition, organic and inorganic cofactors, 3D structure)
…and why?
Purification strategies
•Protein stabilisation:
–Integral membrane Proteins: Solubilisation
–Purification at 4°C: reduces protease activity
–Addition of protease inhibitors: commonly used 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
