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 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

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