protein purification-S2
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Transcript of protein purification-S2
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