Principles and Practice of Protein Purification

Extraction of proteins

Mechanical Lysis for Protein Extraction

Mechanical lysis: disruption of cells using sonication, a

pressure cell, homogenizer, or bead beater.

Mechanical lysis methods are economical and preferable for large-scale preparations.

However, mechanical lysis produces heat, which needs to be controlled. Care should also be taken to avoid foaming, to prevent surface denaturation and oxidation.

Mechanical lysis methods are two types: agitation and liquid shear methods.

•Dispersions •Degassing •Homogenization

Extraction of Recombinant Proteins from Bacteria

Molecular cloning techniques allow high levels of expression of heterologous proteins in bacteria such as E. coli.

They are grown in large scale to obtain the desired amount of protein.

In some cases, bacteria secrete recombinant proteins into the media and thus eliminate the need to lysis the cells. But in most cases, lysis of the bacteria is required to extract the recombinant protein product.

Lysozyme is generally used to cleave the glucosidic linkage of the polysaccharide present in the bacterial cell wall. Detergents, osmotic pressure or other mechanical methods are than employed to disrupt the inner cytoplasmic membrane to release soluble recombinant proteins.

The yeast cell wall can be digested with a variety of enzymes, such as zymolyase, lyticase, and - glucuronidase.

The yeast cell wall contains the carbohydrate glucan, mannoprotein,glycoprotein, and small amounts of chitin.

Enzymatic reactions are usually carried out at room temperature to 37 ℃ in the presence of sulfhydryl reagents in order to

enhance the lysis.

Lysis can be carried out directly, but in most instances spheroplasts are prepared as an intermediate step with these enzymes.

Spheroplasts are then lysed in a variety of ways, such as detergent extraction, homogenization using glass beads, or French press.

Preparation of extracts from yeast

Prior to extraction, solid tissues are cut into small pieces and homogenized with a homogenizer.

The choice of extraction buffer often dictates by the nature of protein that needs to be analyzed.

Proteins whose activities are sensitive to oxidation require extraction in the presence of a reducing agent such as dithiothreitol or mercaptoethanol.

Methods for Working with Protein

Separation methods A. Properties that are used to separate proteins: i. charge ii. hydrophobicity iii. affinity iv. solubility & stability v. molecular weight B. differential centrifugation - S-100 versus S-30 C. precipitation/solubility i. salting in versus salting out solubility of a protein close to its pI versus the effect of salt interacting w/ solvent & not the protein ii. examples of a. ammonium sulfate precipitation b. PEI - poly(ethyleneimine)precipitation

Dialisis

SEPARATION METHODS

ion exchange - cation vs anion

a. strong versus weak (effect of pH)

b. counterion present is important

c. types of gradients and their

• application

• linear, nonlinear, step

Penukar kation Penukar anion

AFFINITY CHROMATOGRAPHY

a. ligand based: • glutathione covalent linked to resin • GST fusion protein b. speciality dyes - Cibracon blue and others c. Immunoaffinity: • epitope tags such as FLAG, V5, etc. d. DNA - general and specific DNAs e. others (example: heparin,

hydroxyapatite)

gel exclusion or gel filtration

a. separation based on size

b. exclusion volume

c. different size limit materials

HIC or hydrophobic interaction chromatography

compare to reverse phase chromatography

types of resin

• cellulose, dextran, agarose, polyacrylamide, perfusion

PROTEIN ANALYSIS WITH SDS-PAGE

POLYACRYLAMIDA

RESULT OF SDS-PAGE

TABEL KEMURNIAN PROTEIN