蛋白质纯化的原理和方法

时间：2025-04-20

纯化原理与方法

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 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

纯化原理与方法

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

纯化原理与方法

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:

纯化原理与方法

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

纯化原理与方法

Max 5% impurities are acceptable In general:Max 4 purification steps No steps with purification factor < 5 No steps with < 30% yield

No steps which last longer than one day and one night Chromatography

Separation material in columns, streamed by buffer →liquid chromatography

HPLC: high pressure/performance liquid chromatography

FPLC: fast protein liquid chromatography

纯化原理与方法

FPLC unit

Separation principles Size:

size exclusion chromatography (= gel filtration, = gel permeation chromatography) Charge

anion or cation exchange chromatography; chromatofocusing Hydrophobicity

hydrophobic interaction chromatography (HIC) Affinity

affinity chromatography Solubility

ammonium sulfate precipitation (non chromatographic, rel. imprecise)

纯化原理与方法

Size exclusion chromatography

Different pore sizes, depending on the size of the proteins Separation is based in diffusion slow flow rate Pressure sensitive materials

Example for a separation by gel filtration

纯化原理与方法

Noteworthy about chromatography Gel filtration:

limited sample volume: 2-3mllimited flow rate gel filtraion takes timedelutionof the sample by a factor of about 3low purificatopn factor: 3-6needs column buffer with high ionic strength: min 0.1 M Ion exchage chromatography:

To remember:Protein binding depents on electrostatic interactions with the column material.Strength of binding depents on pH and ionic strength of the buffer, the pI of the protein and the charge density on the column.

In general:Technical easier than gel filtration: columns could be packed at the FPLC.Sample volume can be multiple times the column volume.Higher flow rates.Purification factor: 3-15Sample gets concentrated. Don’t use charged detergents!

Ion exchage chromatography

Binding behaviour of proteins

pI= isoelectricpoint of the protein the pH value at which the posses no net charge The pI determines the charge of the protein at a given pH

纯化原理与方法

pH > pI negative net charge pH < pI

positive net charge

pIand protein separation on ion exchange columns

纯化原理与方法

But...

pIis not alone responsible for the binding behaviour of proteins

Binding is sometimes influenced by local charges and not by thenet charge of the protein

纯化原理与方法