2. Protein purification Flashcards
What is the 5-step process to express protein in E Coli?
1. Make plasmid containing the gene of interest
2. Transform plasmid in E. coli strain BL21 DE3
3. Grow cells in shaker
4. Adjust temperature and add inducer for expression of protein
5. Harvest cells
What are the 4 consitutents of a plasmid?
- Origin of replication for DNA polymerase
Determines copy number of plasmid (typically low)
- Antibiotic resistance gene
For selection of transformed cells
- Regulated transcription promoter
Under control of an inducible operator (lac operon)
- Multiple cloning site MCS
for insertion gene containing ORF and POI
How do you transform bacterial cells with a plasmid?
How do you culture bacterial cells afterwards?
Heat shock bacteria to make cell wall fragile
or
Electroporation
Grown in rich medium at 37 C
What are the pros and cons of E. coli?
Pros
- Easy
- Fast
- Cheap
Cons
- (mammalian protein in bacteria) might not work
- lacks chaperones, co-factors, PTMs, etc
- Fast translation –> ineffecient folding –> inclusion bodies
What makes P. pastoris (yeast) cells effecient for recombinant protein expression?
How can secretion be achieved in these cells and what is a consequence of this?
What PTM is this cell useful for?
Can use methanol as a sole carbon source
and
Contains the strong promoter for alcohol oxidase (AOX1)
attaching alpha mating factor (aMF) allows for secretion in medium, prevents inclusion body formation and facilitates purification
Useful for secreted, glycosylated protein expression
Describe the Baculovirus-insect cell method of protein expression
What are the downsides to this process?
Why do it then?
Clonal cells isolated from ovarian tissue of moths
These cells are susceptible to baculovirus infection
The large plasmids of these viruses (called Badmids) need to be generated via combination of smaller E. coli plasmids
Creates high-titer virus stocks, highly expressed protein
Expensive and lengthy procedure, but insect cells contain most PTMs found in vertabrates
Describe the process of protein expression in mammalian cells
What are the pros and cons of using mammalian cells?
Use transformed Human embryonic kidney cells (HEK293) or Chinese hamster ovary (CHO) cells
Transfect cells with plasmids for transient expression under strong promoter (eg Cytomegalovirus, CMV)
Generate stable expression cell lines using selectable markers (eg neomycin)
Pros: Ideal environment for mammalian protein expression, contains all necessary factors for proper folding
Cons: Slow, low yields, expensive, requiresw growth factors and specialised media formulation
What are 3 general considerations for the design of a construct?
- Addition of an affinity tag for purification
- eg hexahistidine tag, glutatione-S-transferase, biotin
- N- or C-terminal tag might affect function of protein
- Protease site to remove affinity tag after purification?
- Rhinovirus 3C, TEV, thrombin
- Codon usage: optimize for host expression bias in heterologous case (eg AGG codes for Arg in both humans and E. coli but is rarely seen in bacteria. Use CGU instead)
- Signal peptides for targetting to plasma membrane, secretion?
How can you extract cells from medium?
What are the constituents of resuspension medium?
Do you extract protein from cells?
Low speed centrifugation precipitates cells
Resuspension of cells in medium:
- buffer (pH), isoelectric point (pI) specific to protein so that it is the least soluble possible
- Use HEPES or Tris (org. acid and base)
- Salts (maintain salubility)
- Salting in effect: neutralizing charged group in proteins that can interact witheach other resulting in precipitation
- Salting out effect: enhancing of hydrophobic effect, also precipitation
- Detergents (useful for extracting membrane-bound proteins
- Triton, CHAPS, deoxycholate
- Urea (solubilize protein aggregates
- Protease inhibitors (EDTA) for proteins with no divalent metals
Lyse cells to extract proteins:
- Sonication (bacteria/mammal cells)
- French cell press (bacteria/yeast)
- Freezing-grinding (best method for yeast)
- Lysozyme treatment (Gram -ve bacteria
- Dounce homogenizer (mammalian)
High-speed centrifugation to remove debris
Describe the three consituents for Liquid Chromatography
What are the 4 general steps for LC?
Mobile phase (buffer) in continuous flow
Stationary phase (resin) with desired chemical properites
Differential interactions between proteins and the solid phase allows elution in the mobile phase
- Equilibrate resin with buffer
- Load sample
- Was off contaminants
- Elute Protein of Interest
What are the four main properties exploited for protein purification and how are they used?
What fifth method can purify small-organic molecules or peptides?
BIG question
Affinity Chromatography
- Relies on affinity tags produced during expression
- Glutatione-agarose for GST fusion
- Nickel-NTA-agarose for His6 fusion
- Antibody-resin for antigen binding
- Elution is carried out using high [competitor], or low pH
Size exclusion chromatography
- Porous beads that allow small molecules to pass first, followed by smaller and smaller beads (smaller beads get caught in the pores)
Ion-Exchange chromatography
- Resin can be -vely or +vely charged (R-SO3- or R-NH3+) and binds oppositely charged protein
- Net charge can be modulated with pH
- pH < pI, protein will be +
- pH > pI, protein will be -
- Optimize buffering pH to bind target protein
- “screening” of electrostatic interactions with counterions to elute proteins
- Useul to separate proteins of similar size but different charges
- HIGH SALT WASH
Hydrophobic interaction chromatograpy
- Resin has aromatic/aliphatic hydrophobic groups, react reversibly with proteins
- enhanced by high salt concs
- useful as a polishing step, cheap
- elution down a salt gradient
Reverse phase chromatography
- Used to determine purity of chemical synthesis product
- Mostly used for small org compounds
- Mobile phase: strong acid
- denatures proteins/peptides
What are the two aromatic side chains that significantly absorb light?
What equation can be used to detect the concentration of a protein? What else needs to be known?
Trp and Tyr
Beer-Lambert Law
A = E x B x C
A = absorbance
E = absorption coefficient (n Trp * 5500 + n Tyr * 1490) in 1/M*cm
B is the path length of the UV cell in cm
C is the concentration
How can you analyze protein purity? What does this assay do? How does it work?
SDS-PAGE
Resolves polypeptides vased on molecular weight
SDS detergent confers a constant negative charge/unit mass of protein
mobility down gell depends on charge-mass ratio (constant) and molecular sieving effect (depends on size alone)
Smaller proteins move farther down
