Protein purification:

Protein purification: the basics
Arvind Varsani
http://Protein purification

Reasons for protein purification

•To identify the FUNCTION of a protein
•To identify the STRUCTURE of a protein
•To use the use the purified product –INTERMIDIATE- in downstream reactions / processing
•To produce a COMMERCIAL product

Selection of protein source

•Starting material can be from
–Animal tissue
–Plant material
–Biological fluids (e.g. blood, milk, sera)
RECOMBINANT expression
–Fermentation cultures (yeast, fungi, bacteria)
–Cell cultures (animal cells, plant cells, insect cells)

Important

•Protein in low concentration in natural sources
–Need to induce expression
Or express recombinantly in various expression systems

Key steps in purification

•Release of target protein from starting material
•Removal of solids to leave the protein in the supernatant
•Concentration of the protein
•Removal of contaminants to achieve the desired purity
•Stabilization of the target protein

Three phase purification strategy

The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by 3 major purifications step. The number of steps will depend on the purification strategy, purity requirements and intended use of the protein

Protein analysis

•Tracking protein of interest and determining the yield during purification
–Intended use of protein / source of starting material
•Physical studies e.g. x-ray, NMR, EM
•End product – pharmaceuticals

Analysis of protein purity

•Total protein
•Specific quantification
–Activity assays
–Binding assays
•Detection of impurities
–HPLC
–Gel electrophoresis
•Protein mass spectrometry

Methods for quantification of proteins in solution

Assay method Useful range Comments

NanoOrange assay 100ng/ml to 10ug/ml ·Samples can be read up to six
hours later without any loss in
the sensitivity

·Low protein to protein signal
variability

·Detection not influenced by
reducing agents or nucleic acid

BCA method
(Cu reduction) 0.5ug/ml to 1.5mg.ml ·Samples must be read within
10mins

·Not compatible with reducing
agents

BSA assay (Bicinchoninic acid)

•The first step is a Biuret reaction which reduces Cu+2 to Cu+1
•In the second step BCA forms a complex with Cu+1 which it purple colored and is detectable at 562 nm

Bradford assay (coomassie dye binding)

•Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG) when bound to arginine and aromatic residues
•The anionic (bound form) has absorbance maximum at 595 nm whereas the cationic form (unbound form) has and absorbance maximum at 470 nm

Lowry assay (Cu reduction)

The first step is a Biuret reaction which reduces Cu+2 to Cu+1
The second reaction uses Cu+1 to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). This is detectable in the range of 500 to 750 nm

Absorbance at 280nm

·Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. Higher order structure in the proteins will influence the absorption

Enzyme activity assays

•Continuous (kinetic assays)
–No separation step

Cell disruption / breakage for protein release

•Extraction techniques are selected based on the source of protein (e.g. bacteria, plant, mammalian, intracellular or extra cellular)
•Use procedures that are as gentle as possible. Cell disruption leads to the release of proteolytic enzymes and general acidification
•Selection of an extraction technique often depends on the equipment availability and the scale of operation
•Extractions should be performed quickly, at sub-ambient temperatures in a suitable buffer to maintain pH and ionic strength
•Samples should be clear and free of particles before beginning chromatography

Cell disruption: source variations

•Tissues – variable
•Mammalian cells – easy
•Plant cells – some problems
•Microbial cells – vary, common
•Yeast and fungal cells – more difficult

Cell disruption: methods

1 Chemical / enzymatic
•Cell lysis (osmotic shock and freeze thaw)
•Enzymatic digestion
Blood cells
Mammalian cells
•Fractional precipitation
•Extra cellular proteins

2 Mechanical
•Hand and blade homogenizers
tissue
•Sonicator / disruptors
•Grinding with abrasive
plant/yeast
•Bad beaters / mill
•French press
•micro fluidizer

Lytic enzymes and detergents

•Lysozyme: disrupts bacterial cell walls (hydrolysis of peptidoglycans) leading to cell rupture
–Effective with gram positive bacteria, gram negative generally require pre-treatment with a chelating agent such as EDTA
•Detergents: anionic and non-ionic detergents have been used to permeabilize gram negative cells. Detergents are required for the release of integral membrane proteins.

Simple shear methods

Glass homogenizer (dounce, ten-broeck)

sonicator

•sonicator