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        蛋白质纯化的原理和方法(Protein Purification Principles and Methods)

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        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

        ÆIn 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:ÆFrench Press

        Æ Lysozyme

        Æ Ultrasonic

        French Press

        蛋白质纯化的原理和方法(Protein Purification Principles and Methods)

        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

        Solubilisationof integral membrane proteins

        蛋白质纯化的原理和方法(Protein Purification Principles and Methods)

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