Expressed protein

identification and

purification

Submmited by

Ajay Singh

Ph.D biotechnology

College of biotechnology, DUVASU, Mathura

DNA Vector

• Molecular carriers which carry fragments of DNA

into host cell.

• Usually derived from plasmids, which are small,

circular, double stranded DNA molecule.

Some widely used vectors

1. Plasmid

2. Cosmid

3. Yeast Artificial Chromosomes

4. Phage lambda vectors

5. Bacterial Artificial Chromosomes

6. Human Artificial Chromosomes

7. Transposons etc.

Basic steps to get recombinant protein

• Amplification of gene of interest. ( Using PCR)

• Insert into cloning vector. (Ex: PCR*8)

• Sub cloning into expression vector. (Ex: pKK223-3or PSVK 3)

• Transformation into protein expressing bacteria (Ecoli) or yeast

• Test for identification of recombinant protein.(Western blot or Fluorescence)

• Large scale production.(large scale fermenter)

• Isolation and purification

Isolation of protein:

Initial steps prior to purification:

• Disruption of cells: Osmotic, chemical, Enzymatic,

Mechanical

• Clarification: Centrifugation Filtration

• Concentration: Precipitation Ultra filtration

Differential Centrifugation

Differential Centrifugation

• It’s a procedure in which a homogenate is subjected

to repeated Centrifugations each time increasing the

Centrifugal force

• Separation is based predominantly on particle mass

and size with larger and heavier particles pelleting at

lower Centrifugal fields

• For specific organelle sub cellular fractions

Protein Isolation: Methods for isolation

Lysis buffer

• Used only for bacteria or animal cells

• May cause degradation

• Non machinery

From: bio-ggs.blogspot.com/2009/11/ggs-live-western...

Purification of isolated protein

1. Charge: IEC/IEF

2. Size: size exclusion chromatography

3. Hydrophobicity: Hydrophobic Interaction

Chromatography

4. Ligand specifity: affinity chromatography,

nucleotide and coenzymes resins, phosphoprotein

resins.

5. Avidin biotin matrices

6. Carbohydrate binding

7. Dye resins.

8. Solubility: Precipitation

Separation by expression site

• Selective use of tissues or organelles

• Separation of soluble from membrane proteins

by centrifugation

Separation by size

• Ultrafiltration

• Size exclusion chromatography

• Preparative native gel electrophoresis

Separation by charge

• exchange chromatography

• Isoelectric focusing (as chromatography, in solution

or in gel electrophoresis)

Separation by specific binding sites

• Metal affinity chromatography using natural or

artificial metal binding sites

• Immuno-Chromatography Immunochromatography

using immobilized antibodies

• Magnetic separation using magnetically tagged

antibodies

Protein Purification: Separation by size

exclusion chromatography • Principle: Small proteins can enter more of the

pores in the column material than

• large proteins, so that small proteins migrate

slower

From: elchem.kaist.ac.kr From: http://en.wikipedia.org/wiki

Protein Purification: Separation by size

in native gels• Principle: Small proteins are less retained by the

fibers of the gel than large proteins, so that small

proteins migrate faster

photo of a green gel (Küpper et al., 2003, Funct Plant Biol) From:www.columbia.edu/.../c2005/lectures/lec6_09.html

Size-exclusion chromatography

Absorbance at 280 is used to identify protein-

containing fractions. You can also perform an

enzyme specific assay.

Precipitation

• Proteins tend to precipitate at their isoelectricpoint if the ionic strength of the solution is veryhigh

• First step in protein purification

• Ammonium sulfate and Trichloroacetate (TCA)are the most common salt

Buffer Exchange

• Importance:

Different purification

techniques require the

protein present in

buffers of specific pH

and ionic strengths

Ion

Exchange

Chromatog

raphy

Affinity

Chromatogra

phy

Hydrophobic Interaction

chromatography (HIC)

• It is based on hydrophobic attraction between thestationary phase and the protein molecules

• The stationary phase consists of small non-polargroups ( butyl, octyl or phenyl) attached to ahydrophilic polymer backbone (cross-linked dextranor agarose, for example)

• The sample is loaded in a buffer containing a highconcentration of a non-denaturing salt (frequentlyammonium sulfate)

• The proteins are then eluted as the concentration ofthe salt in the buffer is decreased

Purification of Tagged Recombinant

Proteins• Ni-NTA Agarose To purify recombinant

protein containing polyhistidine (6xHis)sequence

• Streptavidin Agarose To purify biotinylatedrecombinant protein

References

• photo of a green gel (Küpper et al., 2003, FunctPlant Biol)

• From:www.columbia.edu/.../c2005/lectures/lec6_09.html

• From: bio-ggs.blogspot.com/2009/11/ggs-live-western...

• From: http://en.wikipedia.org/wiki• From: elchem.kaist.ac.kr• Willson and walker (2005) “instrumentation”• Harper (2008) “fundamental of biochemistry”

Thanks!