This document discusses methods for expressing and purifying recombinant proteins. It describes commonly used vectors for inserting genes of interest, such as plasmids and artificial chromosomes. The basic steps are outlined as amplifying the gene, inserting it into a cloning vector, subcloning into an expression vector, transforming the vector into a protein-expressing organism, and testing for protein identification. Methods for isolating and purifying the protein include cell disruption, centrifugation, concentration techniques, and differential centrifugation. Common purification methods described are based on separating proteins by charge, size, hydrophobicity, and specific binding sites using techniques like ion exchange chromatography, size exclusion chromatography, and affinity chromatography.

1. Expressed protein
identification and
purification
Submmited by
Ajay Singh
Ph.D biotechnology
College of biotechnology, DUVASU, Mathura

2. DNA Vector
• Molecular carriers which carry fragments of DNA
into host cell.
• Usually derived from plasmids, which are small,
circular, double stranded DNA molecule.

3. 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.

4. 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: pKK223-3
or PSVK 3)
• Transformation into protein expressing bacteria (E
coli) or yeast
• Test for identification of recombinant protein.(
Western blot or Fluorescence)
• Large scale production.(large scale fermenter)
• Isolation and purification

5. Isolation of protein:
Initial steps prior to purification:
• Disruption of cells: Osmotic, chemical, Enzymatic,
Mechanical
• Clarification: Centrifugation Filtration
• Concentration: Precipitation Ultra filtration
Differential Centrifugation

6. 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

7. Protein Isolation: Methods for isolation
Lysis buffer
• Used only for bacteria or animal cells
• May cause degradation
• Non machinery

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

9. 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

10. 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

11. 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

12. 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

13. 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

14. Size-exclusion chromatography
Absorbance at 280 is used to identify protein-
containing fractions. You can also perform an
enzyme specific assay.

15. Precipitation
• Proteins tend to precipitate at their isoelectric
point if the ionic strength of the solution is very
high
• First step in protein purification
• Ammonium sulfate and Trichloroacetate (TCA)
are the most common salt

16. Buffer Exchange
• Importance:
Different purification
techniques require the
protein present in
buffers of specific pH
and ionic strengths

17. Ion
Exchange
Chromatog
raphy

18. Affinity
Chromatogra
phy

19. Hydrophobic Interaction
chromatography (HIC)
• It is based on hydrophobic attraction between the
stationary phase and the protein molecules
• The stationary phase consists of small non-polar
groups ( butyl, octyl or phenyl) attached to a
hydrophilic polymer backbone (cross-linked dextran
or agarose, for example)
• The sample is loaded in a buffer containing a high
concentration of a non-denaturing salt (frequently
ammonium sulfate)
• The proteins are then eluted as the concentration of
the salt in the buffer is decreased

20. Purification of Tagged Recombinant
Proteins
• Ni-NTA Agarose To purify recombinant
protein containing polyhistidine (6xHis)
sequence
• Streptavidin Agarose To purify biotinylated
recombinant protein

21. References
• photo of a green gel (Küpper et al., 2003, Funct
Plant Biol)
• From:www.columbia.edu/.../c2005/lectures/lec
6_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”

22. Thanks!