BMB 407/507 - Section on Practical Methods

Warning: This is a informal summary of some of the material covered in class. These notes are NOT a substitute for attending class or the reading of the review paper listed at the bottom.

Protein Production

• Native sources.


• Bacterial Overexpression:
• Usually in E. coli (on a plasmid under specific antibiotic control)
• Use of the T7 RNA polymerase system (such as pET vectors from Novagen Inc.)
• Affinity tags are often added for purification or increased solubility
• Protein expression is typically inducible using different promoter systems


• Common eukaryotic overexpression systems are:
• Insect cells (e.g. sf9) with baculovirus expression
• Yeast cells (e.g. Pichia pastoris)
• Cultured mammalian cell

Common Affinity/Solubility Tags

• His-tags (approx 20 aa with 6, 8 or 10 his residues)


• GST Tag - Glutathione S-transferase


• Protein A


• CBP (Calmodulin binding peptide)


• CBD (Cellulose binding domain)


• S-tag


• MBP - Maltose binding protein (improves solubility as a fusion protein)


• Trx - Thioredoxin (improves solubility as a N-terminal fusion; usually coupled with another affinity tag)


• NusA - N utilization substance A, which improves solubility as a N-terminal fusion


• IMPACT - chitin binding domain with intein sequences


• TAP tag - Tandem Affinity Purification: CBP + Prot A tags with an intervening TEV cleavage site (see review paper)

Protein Purification

• Cell disruption


• French press
• Freeze/thaw
• Lysozyme and/or other cell disruption cocktails (e.g. BugBuster from Novagen Inc.)


• Initial Steps


• Centrifugation to remove membranes and organelles
• DNA/RNA precipitation or digestion
• Enrichment by salting or desalting


• Column Chromatography


• Affinity chromatography
• IMAC - immobilized metal affinity chromatography (e.g. Ni columns)
• GST columns (immobilized glutathione)
• Antibody affinity (Prot A., etc.)
• Heparin
• Calmodulin
• Chitin
• amylose (for MBP tag)
• Streptavidin


• Ion-exchange chromatography
• Anion exchange (+vely charged beads) - Q or DEAE groups; pH > pI
• Cation exchange (-vely charged beads) - S, SP or CM groups; pH < pI
• Bound proteins can be eluted by either a salt or pH gradient.


• Hydrophobic Interaction Chromatography (HIC)


• Reversed Phase Chromatography (RPC) - usually for small peptides


• Gel filtration chromatography
• Separation by molecular size and shape
• large proteins elute before smaller proteins

Protein Characterization

• Gel electrophoresis - SDS, Native, 2D gels


• Mass Spectroscopy - measures M/Z of ionized particles
• MALDI - Matrix-assisted laser desorption and ionization
• ESI - electrospray ionization
• Use of tryptic fragments for protein identification
• MS/MS or Tandem MS techniques


• Circular Dichroism


• Some techniques for looking at protein-protein interactions and complexes:
• Gel filration chromatography
• Gel shift or filter binding assays
• Ultracentrifugation
• Surface Plasmon Resonance (SPR) - e.g. BIAcore
• Isothermal Titration Calorimetry (ITC)
• TAP tagging
• Immunoprecipitation (IP)
• Epitope tagging and GST pulldowns
• In-vivo approaches such as yeast 2-hybrid

Required Reading

Bauer, A. & Kuster, B. (2003) Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes. Eur. J. Biochem., 270, 570-578. Abstract

Optional Reading & Links

Gavin, A. C. et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415, 141-147. Abstract

Laue, T. (2001) Biophysical studies by ultracentrifugation. Current Opinion in Structural Biology, 11, 579-583. Abstract

Leavitt, S. & Freire, E. (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Current Opinion in Structural Biology, 11, 560-566. Abstract

Notes on the pET system from a Molecular Biology Course at Davidson College. link

Novagen Inc. pET system. link

Pharmacia Protein Purification Handbook - Amersham Biosciences. link

BiaCore/SPR Technology. link