protein
TRANSCRIPT
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!