Lec 1 Crennell Flashcards
What are the 4 main protein properties?
- polypeptide chain
- folded state as purified
- held together by non covalent interactions
- have charge, hydrophobicity, affinity
Why purify a protein?
- must purify an enzyme to look at kinetics
- protein must be 95% pure to look at crystals
- if you want protein as a reagent must be pure or may get side affects
- identify the structure
How pure is pure?
Low purity - low cost, high volume, e.g. industrial bulk enzyme production such as amaylase
Moderate purity - <95% antigen for antibody sequencing,
High purity >95% for structure and characterisation, high cost small volume
Highest 99.9% for therapeutics
Proteins can be naturally sourced. What are the requirements of proteins which are gained in this way, and some of their features?
- the required protein must be abundant and stable
- there must be a wide range of that protein
- plant sources are low abundance
- proteins out of seeds have high storage levels
What must you ensure when gaining a protein from transgenics and in which organisms is it most successful?
- must make sure native protein is knocked out though otherwise will have difficulty separating the two
- most successful in similar organisms
Breif overview of prokaryotic expression of protein, how does it work?
- gene in plasmid under strong promotor control
- expression is induced
Advantages of Prokaryotic expression of protein
- rapid growth
- simple nutrition
Disadvantages of Prokaryotic expression of protein
- No post-translational modification
- Often insoluble
Issues with using prokaryotes for protein expression
- post translational modifications wont happen in bacteria that happen in eukaryotes, e.g. sugars not added etc
Eukaryotic protein expression (Yeast) advantages
- Good growth rates
- simple
- cheap
- genetics understood
Eukaryotic protein expression (Yeast) disadvantages
- misfolding
- hyperglycosylation
- not high cell density
Mammalian recombinant expression advantages
- correct fold
- secreted
- no molecular weight limit
Mammalian recombinant expression disadvantages
- low yield
- expensive
- 2 months
- fragile cells
- infections impurities
- complicated media
Mammalian transgenic expression (goat milk etc) advantages
- yield relatively good
- secreted in milk
- correct fold
- only expressed in milk
Mammalian transgenic expression (goat milk etc) disadvantages
- must make sure what you’re expressing isn’t toxic
- fragile cells
- infectious impurities
- cost is high
Why is it better to have organisms close together in expression?
- will express better but it is expensive in some cases so if yo can use bacteria then do
How does extraction of protein homogenisation (natural sources)
in MAMMALIAN work?
Mammalian tissues - must break open tissues to get cells out, e.g. cut into small pieces, blend, keep cold, isotonic buffer to mimic salts in cell.
- ease of this dependent on tissue type e.g. lungs are difficult
How does extraction of protein homogenisation (natural sources)
in PLANTS work?
- Cell walls harder
- Blend for fragile tissues e.g. leaf
- grind with sand and salt in pestle and morter for fibrous tissues e.g. stem
( freeze in N2 if protein stable)
Cell lysis used to extraction proteins. What is cell lysis?
- breaking of plasma membrane (cell death)
Why must the release of proteases in extraction of protein Cell lysis be stopped?
proteases will break down everything even your desired protein
What is the order of ease in Extraction of protein via Cell lysis?
- Eukaryotic easiest
- bacteria tricky as peptidoglycan wall
- Plants hardest as heavy cellulose wall
Extraction of protein Cell lysis - PHYSCIAL CELL DISRUPTION
MECHANICAL, how does it work?
- Rotating blades e.g. waring blender
- grinding in pestle and mortar (with acid sand or N2)
- vortexing with glass beads
Extraction of protein Cell lysis - PHYSCIAL CELL DISRUPTION
LIQUID SHEAR, how does it work?
- French press homogenizer
- suspension forced through narrow space (pressure release bursts cells)
- only small volumes/difficult to clean
Extraction of protein Cell lysis - PHYSCIAL CELL DISRUPTION
SONICATION, how does it work?
- high frequency sound waves to break open cells
- pressure felt by solution, as sound wave moves away solution can expand and bubbles form, they get bigger then compressed as sound wave comes back again and cells burst
- oscillating metal probes
- shear cells (implosion: heat and pressure)
