protein purification principles and methods：蛋白质纯化的原理和方法.pdfVIP

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 and 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 • Peripheral 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 Solubilisation of integral membrane proteins • Solubilisation of 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) Na-Deoxycholate: