PPT On Protein Purification and Analysis

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Protein Purification and Analysis Presentation Transcript:
1.Protein Purification and Analysis

2.Protein Purification and Analysis
    Numbers of genes:
Humans    ~40,000 genes
Yeast        ~6000 genes
Bacteria    ~3000 genes
    Solubility of proteins important for purification:
60-80% soluble, 20-40% membrane
Some proteins expressed at high levels (collagen, hemoglobin)
Some proteins expressed at low levels (repressors, signaling)
Fibrous proteins - structural (collagen, elastin, keratin)
Globular proteins - structure and/or function (actin, enzymes)

3.Steps of purification and analysis
  (1)  Choose protein to purify
  (2)   Choose source (natural or expressed)
  (3)   Soluble in aqueous solution?? (problem     with       membrane proteins)
  (4)   Stability
  (5)   Purify
  (6)   Study (activity, structure, mechanism of        action, etc.)

4.Protein Purification and Analysis
(1) Choose protein to purify -
(2) Choose source (natural or expressed)
Source of protein for study
Early biochemistry (1970’s)
      utilized proteins that were abundant from natural sources
      (myoglobin, lysozyme, hexokinase)
 Middle biochemistry (1980’s to mid 1990’s)
      isolated small amounts of proteins, get gene, express and  
      purify from bacteria, yeast, insect cells, mammalian cells
Now (2000s)
     get gene from library based on homology
     choose gene and express and study it
Still problems with:
membrane proteins and solubility

5.Protein Purification and Analysis
 (2) Choose source (natural or expressed)
Break open cells by destroying membranes and releasing cytosolic protein mix - crude extract
If nuclear or membrane protein - more work!
(3) Soluble in aqueous solution?? (problem with membrane proteins)
(4) Stability (perform purification/analyses in cold)
(5) Purify
Separate proteins using fractionation based on physical characteristic:
1. solubility
2. electrical charge
3. size + shape 
4. affinity for other molecules 
5. polarity

6.Chromatography
        Important steps:
   1. Pack column - Column is packed with material (resin)
     that can absorb molecules based on
      some property (charge, size,  
      binding affinity, etc.)
   2. Wash column – Molecules washed
      through the column with buffer
   3. Collect fractions - Fractions are taken, at some point your molecule will elute

7.Ion exchange chromatography
Separate by charge
Elute protein
    Increase salt or pH to
    elute protein of interest

8.Protein Purification and Analysis

9.Protein Purification and Analysis
Additional Chromatography info
HPLC (high-performance liquid chromatography)
Column can be:
hydrophobic, (+) or (-) charged, stereospecific, etc.
Resin needs to have incompressible beads
high pressure pumps speed the movement of proteins down the column
HPLC limits protein band spreading - increase resolution

10.Gel Electrophoresis

11.Electrophoresis
Separation of proteins, nucleic acids, etc. by size, shape, charge
Proteins migrate based on their charge-to-mass ratio
Proteins visualized (radioactivity or staining)
Use gels made of crosslinked polymer (polyacrylamide) or solidified agarose

12.SDS Gel Electrophoresis
Used to estimate purity and molecular weight, separate proteins by size
Denature protein by adding SDS (then separate by size only)

13.Isoelectric focusing gel electrophoresis
determine the isoelectric point (pI) of a protein
separates proteins until they reach the pH that matches their pI (net charge is zero)

14.Separate proteins by size or density
Differential centrifugation - separates large from small particles
Isopycnic (sucrose-density) centrifugation - separates particles of different densities

15.Protein Sequencing
Function of protein depends on its amino acid sequence
Proteins with different functions always have different sequences
Changing just 1 amino acid can make a protein defective
Functionally similar proteins from different species have similar sequences