Lecture 1 Flashcards
What are the reasons we isolate a protein?
- identify function, specificity and kinetics
- identify structure
- use as a reagent
- diagnostic
How pure do we need proteins?
- defined by the end use. i.e therapeutic 99.9% pure vs industrial bulk enzyme
What are the two protein sources?
natural vs recombinant
What are two requirements of obtaining proteins from natural sources?
- must be adundant and stable
- source must be readily available
What are the disadvantages of plant proteins sources?
- can oxidise
- seeds have high level of storage proteins
- fruits may have to ripen for their proteins to be ready
What is the advantage of natural sourced proteins?
in vivo
What are examples of recombinant protein sources?
- bacteria ,yeast , insect , plant cells
- transgenic animals
What is an advantage of recombinant sources?
yields a lot higher
What are advantages of prokaryotic protein expression?
- rapid growth
- simple nutrition
What are disadvantages of prokaryotic protein expression?
- no post- translation modification
- often insoluble
What are the advantages of yeast (eukaryotic) protein expression?
- good growth
- simple to grow
- genetics understood well
What are the disadvantages of yeast (eukaryotic) protein expression?
- misfolding
hyperglycosylation
-not high cell density
What are the pros and cons of pichia pastoris (eukaryotic)protein expression
pros
- higher cell density
- grows on methanol
cons
- still misfolds
How do you transfect insect cells with the gene?
by a virus
What are the advantages of mammalian recombinant protein expression?
- correct fold
- recreated into the cytosol
- no weight limit
What are the disadvantages of mammalian recombinant protein expression?
- low yields
- fragile cells
- infectious impurities
- complicated media
what are the advantages of mammalian transgenic protein expression?
- high yield
- secreted ( via milk for example)
- correct fold
what are the disadvantages of mammalian transgenic protein expression?
- canbe toxic to the animal
- fragile cells
- infectious impurities
- high cost
How do you homogenise mammalian tissue?
- cut up into small pieces, then blender, high salt concentration and keep cool
How do you homogenise fragile plant tissues?
blend then grind
How do you homogenise fibrous plant sources?
grind with sand or place in liquid nitrogen
What is the point of homogenisation?
break open tissues to get the cells out
What are the two methods to lyse cells?
- physical
- non - mechanical methods
Examples of physical mechanical cell disruption
- blender
- Grind in nitrogen
- grind with acid washed sand
- compression and expansion
- ultrasonicator
Examples of liquid shear physical cell disruption
- French press homogeniser
- suspension forced through narrow space
- only small volumes , difficult to clean
What is sonication?
- physical disruption of cells
How does sonication work?
As sound wave moves away from solution , solution expand, bubbles form. Bubbles then get compressed as the wave comes back . Eventually the bubbles collapse this pressure breaks open the cells. Applies heat and pressure
What are the drawback of mechanical methods?
- heat (denaturation)
- hearing ( need sonic cabinets)
What are the 4 non - mechanical lysis techniques?
- osmotic lysis
- freeze
- detergent solubilisation
- lytic enzyme damage cell walls
What is subcellular fractionation?
Cell fractionation is the process used to separate cellular components while preserving individual functions of each component.
What are the 4 steps for protein extraction?
- Homogenisation
- Cell lysis
- Subcellular fractionation
- purification
What is a suitable cell lysis method for animal tissue?
Blender
What is a suitable cell lysis method for animal cell culture?
physical
- blender
- compression/expansion
Non - mechanical
- osmotic shock
- freeze thaw
- lytic enzyme
What is a suitable cell lysis method for plant tissue?
Physical
- Blender
- Grind in liquid N2
- Grind with acid washed sand
What is a suitable cell lysis method for plant cell culture?
Physical
- Blender
- Grind in liquid N2
- Grind with acid washed sand
- compression/ expansion
- ultrasonicator
Non - mechanical
- Freeze
what is a suitable cell lysis method for bacteria?
Physical
- -Grind in liquid N2
- Grind with acid washed sand
- compression/ expansion
- ultrasonicator
Non mechanical
- Freeze
- detergent
what is a suitable cell lysis method for yeast?
- grind in liquid N2
- ultrasonicator
- Freeze
- enzyme
What are drawbacks of mechanical methods?
- heat
- denaturation
- hearing
