biotechnology field

7/30/2019 biotechnology field

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Derived from the Greek word Chroma meaning

color.

Chromatography provides a way to identify unknown

compounds and separate mixture.


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Definition

Separation technique based on the differentinteractions of compounds with two phases, a mobilephase and a stationary phase, as the compounds travelthrough a supporting medium.

Components mobile phase: a solvent that flows through the

supporting medium.

stationary phase: a layer or coating on the supporting

medium that interacts with the analytes. supporting medium: a solid surface on which the

stationary phase is bound or coated.


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Affinity Chromatography (AF) is a technique

enabling purification of a biomolecule with respect to

biological function or individual chemical structure.

The molecule of interest will have a well known and

defined property which can be separate from complex

mixtures in single process. The target molecule becoming trapped on a solid or

stationary phase or medium.

The other molecule in solution will not become

trapped as they do not possess this property. The solid medium can be removed from the mixture,

washed and the target molecule released from the

entrapment in a process known as elution.


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Liquid chromatography technique that separates biomolecules

according to hydrophobicity.

Used to separate proteins with difference in hydrophobicity

Principle

Separation of substances is based on their varying strength of interaction

with hydrophobic groups attached to an uncharged gel matrix.

Hydrophobic groups on proteins are sufficiently exposed to bind to the

hydrophobic groups on the matrix.

How is this achieved?


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General Concept:

o Salt -promoted adsorption.

Hydrophobic groups on gel matrix and soluble

proteins are shielded by water molecules. To exposethese hydrophobic regions, water must be removed,

and this can be achieved by adding ammonium sulfate.


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Exposed hydrophobic regions of proteins and

ligand group will interact, leading to bindingof protein to ligand.


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To release the bound protein from the ligand,dissociation can be achieved by eluting bound

protein with medium of low ionic strength


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HIC media are composed ofligands containing alkyl oraryl groups.

The medium is packed into a column to form apacked

bed. The bed is then equilibrated with buffer that fills the

pores of the matrix and the space in between theparticles

Interaction between the protein and the medium ispromoted by moderately high salt concentrations

The type of salt and the concentration required in thestart buffer are selected


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When sample loading is completed and the column has

been washed so that all non-bound proteins have passedthrough (i.e., the UV signal has returned to baseline),

conditions are altered to begin elution.

Proteins are eluted by decreasing the salt concentration

in the elution buffer.

As the level of salt decreases those proteins with the

lowest hydrophobicity begin to elute from the column.

By controlling changes in salt concentration using

gradients, proteins are eluted differentially in a

purified, concentrated form.


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Those proteins with the highest degree of

hydrophobicity will be most strongly retained and will

be eluted last. A wash step in a salt-free buffer removes most tightly

bound proteins at the end of an elution.

If the hydrophobicity of the medium and the proteins in

the sample have been judged correctly, all proteins willbe eluted by this stage.

The column is then re-equilibrated in start buffer before

applying more samples in the next run.


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Gradient ionSubstances to be

separated

Starting buffer

c0unter-ions


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IEC is a process that allows the separation of ions andpolar molecule based on the charge properties of the

molecules.

It can be used for almost any kind of charged molecule

including large proteins, small nucleotides and amino acids.

The solution to be injected is usually called a sample, and

the individually separated components are called analytes.

It is often used in protein purification.

Ion exchange chromatography retains analyte molecules

based on (ionic) interactions.

The stationary phase surface displays ionic functional

groups that interact with analyte ions of opposite charge


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The surface charge of the solutes (proteins, nucleic

acids, endotoxin) which bind will be net negative.Commonly used anion exchange resins are Q-resin, a

Quaternary amine; and DEAE resin,

DiEthylAminoEthane (see figure below). AEC is often

used as a primary chromatography.


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The surface charge of the solutes (proteins, nucleicacids, endotoxin) which bind will be net positive.

Commonly used cation exchange resins are S-resin,

sulfate derivatives; and CM resins, carboxylate derived

ions.


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A sample is introduced, either manually or with an

autosampler. A buffered aqueous solution known as the mobile phase

carries the sample from the loop onto a column that

contains some form of stationary phase material which is

typically a resin or gel matrix consisting of agarose or

cellulose beads with covalently bonded charged

functional groups.

The target analytes (anions or cations) are retained on

the stationary phase but can be eluted by increasing the

concentration of a similarly charged species that will

displace the analyte ions from the stationary phase.


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Fast separation technique It is a non-denaturing

technique that can be used

at all stages and scales of

purification

Large volume of sample

can be loaded

Simple and very effectivepurification tool

It can serve as aconcentrating step. A large

volume of dilute sample

can be applied to a media,

and the adsorbed protein

subsequently eluted in asmaller volume

Sample with high ionicstrength can be used

High selectivity It offers high selectivity,

in that it can resolve

molecules with small

differences in charge

Sample eluted with low

salt


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Expensive ligands pH May require high volume of eluent

Leakage of ligand Salt used Usually a relatively slow process,but rapid selective elutionprocesses are known

Degradation of the solid support Buffer type Requires narrow pH control.

Limited lifetime-Resins seem tohave finite lifetime, especiallysubstrate analog resins. They loseeffectiveness with time.

Temperature

Non-specific adsorption Each of these must be carefullycontrolled to achievereproducible separation but alsorepresent an opportunity forincreased selectivity.

Relatively low productivity


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Widely application:

Affinity chromatography is widely used in the pharmaceutical industry topurify and extract molecules of interest from complex mixtures.

Separation of native from denatured forms of proteins

Removal of small amount of biomaterial from large amounts of

contaminants

Purify and concentrate a molecule from a mixture in solution, even at verylow concentrations.

Reduce the amount of a substance in a mixture.

Purify and concentrate an enzyme solution.


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Softening of water Demineralization of water

Purification of solutions free from ionic

impurities Separation of inorganic ions

Separation of sugars, amino acids


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Crude purification of human autotaxin.

Purification of recombinant HIV reverse transcriptase

Purification of mammalian transcription factors

Micropurification of GTPase activating protein


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Purification of recombinant nucleocapsid (NP)protein of

Newcastle disease virus (NDV) from unclarified

feedstock using expanded bed adsorption

chromatography.

Source: Protein Expression and Purification journal

(Tan et al., 2006)


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NP protein is the major protein in Newcastle disease

virus (NDV) and is highly immunogenic.

It has successfully tested on ELISA for detecting the

antibody (Timani, 2004). It can be used in immunodiagnostic kit for NDV

detection in infected chicken serum.


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To develop a simple and rapid technique to recover NPprotein from unclarifiedE. coli using IMA-EBAC.

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**Immobilized metal affinity - expanded bed adsorptionchromatography


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IMAC-EBAC resulted in a 31% adsorption and 9.6% recovery of

NP protein, improve compared to the 1.3% final yield obtained from

the multistep conventional methods.


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NP protein has been efficiently purified fromunclarified feedstock by using IMA-EBAC without the

need of any clarification and concentration steps.

The purity of the NP protein recovered is reasonably

high (about 70%)


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Approximately, 38 mg or

about 31% of bound NP protein

has been eluted from the

adsorbent by elution buffer with

high concentration (350 mM).


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A two-step elution protocol was used to elute NPprotein from the adsorbent containing

1. Elution buffer, (50 mM) can eliminate high

amount of contaminating proteins.

2. Elution buffer, (350) efficient to elutes large

amount of NP protein from the adsorbent.

IMA-EBAC has reduced the downstreamprocessing time.

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