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