week 3: protein purification Flashcards
section 4 (32 cards)
What are common protein sources for purification?
Recombinant proteins (bacteria, mammalian cells)
Endogenous proteins (tissues with high expression)
Why are E. coli (BL21 DE3) commonly used for protein expression?
Fast growth
High yield
Low cost
Easy transformation with plasmids
What are limitations of bacterial expression?
Incorrect folding (inclusion bodies)
Lack of post-translational modifications
What methods are used for bacterial cell lysis?
Freeze-thaw
Detergents (e.g. Triton X-100)
Sonication (ultrasound)
What must be protected during lysis?
Protein functionality (avoid denaturation)
What properties of proteins can be exploited for purification?
Size
Charge
Hydrophobicity
Binding affinity
Biological activity
What is the principle of affinity chromatography?
Specific interaction between the protein and a ligand attached to beads (e.g. nickel beads for His-tags)
How are His-tagged proteins purified?
Bind to Ni²⁺ beads → Wash → Elute with imidazole.
Other tags used for purification?
Biotin (binds streptavidin)
GFP (used with GFP-TRAP)
FLAG tags
What is ammonium sulfate precipitation (“salting out”)?
High salt displaces water, causing protein precipitation.
Why use ammonium sulfate?
Cheap, highly soluble, gentle on proteins (retains function).
How is salt removed after precipitation?
Dialysis
Gel filtration
Diafiltration
What does IEC separate by? (Ion Exchange Chromatography)
Protein charge (positive or negative)
Key resins used in IEC?
Cation exchanger (negatively charged resin: CM-cellulose, S-Sepharose)
Anion exchanger (positively charged resin: DEAE, Q-Sepharose)
What is the isoelectric point (pI)?
The pH where protein has no net charge (least soluble).
How are proteins eluted in IEC?
Increasing salt concentration competes for ionic interactions.
What is GFC also called? (Gel Filtration Chromatography)
Size exclusion chromatography.
What does GFC separate by?
Protein size and shape.
How do large proteins behave in GFC?
Elute first (don’t enter pores).
Why is GFC suitable after ammonium sulfate precipitation?
High salt doesn’t interfere with separation.
What does HIC separate by? (Hydrophobic Interaction Chromatography)
Hydrophobicity of protein surfaces.
What conditions enhance hydrophobic interactions?
High salt concentrations (e.g. ammonium sulfate).
How are proteins eluted in HIC?
By decreasing salt concentration.
How does IEF work? (Isoelectric Focusing)
Proteins migrate in pH gradient until pH = pI.
