Protein Purification

1
Protein Purification

• Why Purify- Arthur Kornberg Handout
• Strategy
• Starting materials,
• Capture, Intermediate Purification, polishing
• Assays, quantitation and documentation

2
Protein Purification Principles I (From
APBiotechBiochemistry 472/578 Resources)

• Define objectives
• for purity, activity and quantity required of
  final product to avoid over or under developing a
  method
• Define properties of target protein and critical
  impurities
• to simplify technique selection and optimisation
• Develop analytical assays
• for fast detection of protein activity/recovery
  and to work efficiently
• Remove damaging contaminants early
• for example, proteases

3
Protein Purification Principles II

• Use a different technique at each step
• to take advantage of sample characteristics which
  can be used for separation (size, charge,
  hydrophobicity, ligand specificity)
• Minimize sample handling at every stage
• to avoid lengthy procedures which risk losing
  activity/reducing recovery
• Minimize use of additives
• additives may need to be removed in an extra
  purification step or may interfere with activity
  assays
• Minimize number of steps - KEEP IT SIMPLE!
• extra steps reduce yield and increase time,
  combine steps logically

4
Starting materials

• Natural source or artificial expression system
• Host for expression,
• Bacteria, yeast, plants, transgenic animals
• Abundance, contaminants
• Lysis and clarification procedures
• Native or denaturing conditions
• Subcellular fractionation
• Selective precipitation
• PEI or Streptomycin Sulfate for RNA and DNA
• Ammonium Sulfate for Proteins

5
Capture

• Quickly remove most damaging contaminants
• Concentrate, adsorption methods
• Ion Exchange most general
• Affinity chromatography can combine capture,
  intermediate and polishing steps
• This step should remove most unwanted contaminants

6
Intermediate purification

• Use a different technique
• Affinity chromatography, Hydrophobic interaction
  chromatography
• Starting conditions are specific for each
  technique
• Buffer must be compatible with adsorption
• Can change buffer by dialysis or desalting by GFC
• Adsorption techniques result in small volume
  concentrated sample

7
Polishing

• Final removal of trace contaminants
• Often size exclusion chromatography
• Buffer exchange is a part of the process
• Sample volume always increases need to start with
  a concentrated sample
• Sample can be concentrated by
• Precipitation (selective or nonselective)
• Ultrafiltration (dialysis under pressure)

8
Assays, Quantitation and Documentation

• Assay enzyme activity at every step
• Contaminants at early stages can mask or inhibit
  activity
• Inactivation can occur at high temperatures,
  because of proteolysis, oxidation, aggregation,
  etc.
• Assay total protein
• Run an SDS gel to visualize specific contaminants
• Specific activity is defined as units of
  enzymatic activity per unit of total protein -
• Yeild can be defined in terms of total protein
  mass, and total enzyme units
• Goal is a high yield and high specific activity.

9
Expt 5 Purification of Alkaline Phosphatase (AP)

• Periplasmic Protein in E. coli
• The space between the rigid peptidoglycan cell
  wall and the osmotically sensitive plasma
  membrane
• Phosphate scavenger
• Liberates Pi from a variety of substrates
• Induced by phosphate starvation
• Used to remove terminal phosphates for selective
  DNA ligation reactions
• Heat stable, Zn enzyme
• Will be used for enzyme kinetics in Experiment 6.

10
Text Book Purification Overview (N B 175-195)

• 1. Lysozyme treatment to release periplasmic
  proteins
• Centrifugation to separate soluble AP from cells
• Dialysis to remove starting buffer (overnight)
• 2. Heat treatment to precipitate weaker proteins
• Centrifugation to separate soluble AP from
  insoluble PPT
• Ammonium sulfate to concentrate proteins/remove
  non protein contaminants
• Dialysis to remove ammonium sulfate (O/N)
• 3. Anion exchange (DEAE) chromatography
• Step elution with 0.125M Salt
• 4. SDS Page to quantify the proteins in each
  fraction

11
Starting material

• E. coli cells starved for phosphate
• Sucrose shrinks the plasma membrane reduces
  turgor pressure
• Lysozyme cleave glycosidic linkages in cell wall
• DNAse reduces viscosity from inadvertantly lysed
  cells
• Left with AP, DNAse, Lysozyme, Sucrose other
  periplasmic and cytoplasmic contaminants

12
Alternative strategy

• Osmotic shock used to liberate periplasmic
  proteins
• Many fewer proteins in periplasm than cytoplasm
• Sucrose draws water from cytoplasm, shrinks inner
  membrane
• EDTA permeabilizes cell wall
• Transfer to low osmotic strength buffer causes
  the inner membrane to slam into the cell wall and
  force out periplasmic proteins
• Periplasmic proteins, no lysozyme, no DNAase, not
  much sucrose
• Periplasm rotococols in Biochemistry 472/578
  Resources from Novagen

13
Assays

• Enzymatic assays
• PNPP is hydrolyzed to PNP and Pi
• Fixed time assay
• Mix enzyme and substrate, react for a fixed time,
  s
• top the reaction with a strong base,
• read the concentration of PNP at pHgt10
• Continuous assay
• Monitor PNP production directly in the spec at ph
  8
• Bradford Assays for total protein
• SDS page for the distribution of proteins by
  size.