Extraction and Purification

Proteins/enzymes
• All living beings like animal ,plant, and microbs etc
contain a lot of various types of protein or enzymes.
• Theses Enzymes can be divided into two groups:
intracellular and extracellular.
• Enzymes formed and retained in the cell are known as
intracellular enzymes,
• which occur in the cytoplasm, organelles or the nucleus.
Examples of intracellular enzyme are
• DNA polymerase, RNA polymerase and ATP synthetase.
Glycolysis enzyme, TCA enzyme and in
photosynthetic pathway enzyme. The lysosome
contains many enzymes that are mainly responsible for
destroying old cells.
• Extracellular enzymes are produced in the cell
then packed and secreted from the cell,
Extracellular enzymes catalyze their reactions
outside the cell. Most digestive enzymes are
extracellular enzymes. For example, amylase,
cellulase and zymase.
• Steps involved in purification of a protein or
enzyme:
• Extraction
• Salt precipitation using (NH4)2SO4
• Dialysis
• Size exclusion chromatography (also called Gel
filtration or Molecular sieving)
• Ion exchange chromatography
• Affinity chromatography
• Lipid nanoparticle solubilize the protein
• Oxidative modifications of proteins can
change their physical and chemical
properties, including conformation,
structure, solubility, susceptibility to
proteolysis, and enzyme activities
• Surfactant substance such as a detergent
that, when added to a liquid, reduces its
surface tension, thereby increasing its
spreading and wetting properties In the
dyeing of textiles, surfactants help the dye
penetrate the fabric evenly.
 Chemical method
1.Alkali method .
Use NaoH, pot hydroxide
2.Acid method.
TCA, sulphoselicylic acid,Hcl,H2SO4 etc
3. Detergent method
Anionic (dodicyle sulphate)
Non anionic( triton y-100-200-50) tween 10-80
4.Enzyme method (Lysozyme)
 Separation of large parts
• Once cells are disrupted, either we have to
collect parts we need or remove unwanted parts.
– FILTRATION
• Cheese cloth: mainly removes materials that are not
homogenized
• Screens: calibrated wire screens can separate broken and
non-broken eukaryotic cells
• Filters: Variety of filters are available from 0.22µm to some
that will allow proteins molecules less than 3000 to pass are
available. Problem – plugging can be reduced by tangential
flow.
 Separation of large particles
• Centrifugation: Accelerated sedimentation.
Sedimentation rate depends on several
factors.
– Force pushing down due to gravity F= mg
– Opposing forces
• Buoyancy density of the particle and the buoyant
force or the density of the solvent
• Frictional force depends on shape of the particles
and the material with which it is going.
Precipitation of proteins
 Solubility in water
• Proteins usually carry charge due to
hydrophilic amino acids and terminal
amine and acid groups. Because they
have hydrophilic amino acids on their
surfaces that attract water molecules and
interact with them, proteins are soluble in
water solutions.
 Protein Precipitation

• "Salting Out" when enough salt has been added, proteins precipitate
• cold prevents denaturation
• collect by filtration or centrifugation
• redissolved in solution using a buffer with low salt content.
• works best with divalent anions like sulfate, especially ammonium sulfate which is highly soluble at ice temperatures
 dialysis
• dialysis is the process of
separating molecules in solution by the difference in their
rates of diffusion through a semipermeable membrane,
such as dialysis tubing.
• most common application of dialysis is for the removal of
unwanted small molecules such as salts, reducing
agents, or dyes from larger macromolecules such
as proteins, DNA, or polysaccharides. Dialysis is also
commonly used for buffer exchange and drug binding
studies.
 Dialysis Membranes and MWC
• Dialysis membranes are produced and characterized according
to molecular-weight cutoff (MWCO) limits.
• While membranes with MWCOs ranging from 1-1,000,000 kDa are
commercially available, membranes with MWCOs near 10 kDa are
most commonly used.
• The MWCO of a membrane is the result of the number and average
size of the pores created during production of the dialysis
membrane.
• The MWCO typically refers to the smallest average molecular mass
of a standard molecule that will not effectively diffuse across the
membrane during extended dialysis.
• Thus, a dialysis membrane with a 10K MWCO will generally retain
greater than 90% of a protein having a molecular mass of at least
10kDa.
 chromatography
 The process or technique of separating molecules or
components in a mixture solution (gas or liquid)
according to the differential absorption and elution
 Invented in 1906 by the Russian botanist Mikhail Tsvet
 Chromatography is the physical separation of a mixture
into its individual components.
 Used in qualitative and quantitative analysis of
biological and chemical substances
• Types of chromatography
• Column chromatography
• Ion-exchange chromatography
• High-pressure liquid chromatography (HPLC)
 Column chromatography

• Since proteins have difference characteristic features as size,

shape, solubilty.net charge, stationary phase used, and binding
capacity, each one of these characteristic components can be
purified using chromatographic methods.
• Among these methods, most frequently column
chromatography is applied. This technique is used for the
purification of biomolecules. On a column (stationary phase)
firstly the sample to be separated, then wash buffer (mobile
phase) are applied Their flow through inside column material
placed on a fiberglass support is ensured. The samples are
accumulated at the bottom of the device in a tme-, and
volume-dependent manner
 procedure
• In column chromatography a glass column
of 80-90cm in length and 2-2.5 diameters.
• Wash the column carefully and remove the
contamination.
• About 10g of gel soaked in 100-150ml
buffer overnight for swelling gel.
• Gel is selected on the basis of protein
separation
 Fractionation Range
sephadexGel Type Globular
Dextrans
Proteins
G-10 ≤700 ≤700
G-15 ≤1500 ≤1500
G-25 1000–5000 100–5,000
G-50 1500–30,000 500–10,000
G-75 3000–80,000 1000–50,000
G-75 SF 3000–70,000 1000–50,000
G-100 4000–150,000 1000–100,000
G-100 SF 4000–100,000 1000–100,000
G-150 5000–300,000 1000–150,000
G-150 SF 5000–150,000 1000–150,000
G-200 5000–600,000 1000–200,000
G-200 SF 5000–250,000 1000–200,000
Gel Particle size Fractionation
range, range limits in
hydrated Daltons
beads (µm)
Bio-Gel P-2 45-90 100-1,800
Bio-Gel P-2 Fine < 45 100-1,800

Bio-Gel P-4 Gel, Fine 45-90 800-4,000

Bio-Gel P-6 Gel, Fine 45-90 1,000-6,000

Bio-Gel P-10 Gel, Fine 45-90 1,500-20,000

Bio-Gel P-30 Gel, Fine 45-90 2,500-40,000

Bio-Gel P-60 Gel, Fine 45-90 3,000-60,000

Bio-Gel P-100 Gel, Fine 45-90 5,000-100,000

 Ion Exchange Chromatography
Ion exchange chromatography -- is a separation based on
charge
Used for almost any kind of charged molecules --- large
proteins, small nucleotides and amino acids
Ion-exchange chromatography preserves analyte
molecules on the column based on ionic interactions
Mobile phage – buffer, pH and salt concentration---
opposite charged solute ions attracted to the stationary
phage by electrostatic force
Stationary phage– resin is used to covalently attach
anions or cations onto it
Cation exchange chromatography
---positively charged molecules are attracted to a
negatively charged solid support. Commonly used
cation exchange resins are S-resin, sulfate
derivatives; and CM resins, carboxylate derived
ions
Anion exchange chromatography
---negatively charged molecules is attracted to a
positively charged solid support. Commonly used
anion exchange resins are Q-resin, a Quaternary
amine; and DEAE resin, DiEthylAminoEthane
anion exchangers Cation exchanger

• Anion exchanger • Cation exchanger

• Aminoethyl (AE-) Carboxymethyl (CM)
Diethylaminoethyl sephadex
(DEAE-)sephadex A-50 • Phospho
• Quaternary aminoethyl • Sulphopropyl (SP-) 9
(QAE-)