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BB10186: Proteins purification and characterisation > Lecture 1 > Flashcards

Lecture 1 Flashcards

1
Q

What are the reasons we isolate a protein?

A
  • identify function, specificity and kinetics
  • identify structure
  • use as a reagent
  • diagnostic
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2
Q

How pure do we need proteins?

A
  • defined by the end use. i.e therapeutic 99.9% pure vs industrial bulk enzyme
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3
Q

What are the two protein sources?

A

natural vs recombinant

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4
Q

What are two requirements of obtaining proteins from natural sources?

A
  • must be adundant and stable

- source must be readily available

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5
Q

What are the disadvantages of plant proteins sources?

A
  • can oxidise
  • seeds have high level of storage proteins
  • fruits may have to ripen for their proteins to be ready
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6
Q

What is the advantage of natural sourced proteins?

A

in vivo

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7
Q

What are examples of recombinant protein sources?

A
  • bacteria ,yeast , insect , plant cells

- transgenic animals

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8
Q

What is an advantage of recombinant sources?

A

yields a lot higher

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9
Q

What are advantages of prokaryotic protein expression?

A
  • rapid growth

- simple nutrition

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10
Q

What are disadvantages of prokaryotic protein expression?

A
  • no post- translation modification

- often insoluble

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11
Q

What are the advantages of yeast (eukaryotic) protein expression?

A
  • good growth
  • simple to grow
  • genetics understood well
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12
Q

What are the disadvantages of yeast (eukaryotic) protein expression?

A
  • misfolding
    hyperglycosylation
    -not high cell density
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13
Q

What are the pros and cons of pichia pastoris (eukaryotic)protein expression

A

pros

  • higher cell density
  • grows on methanol

cons
- still misfolds

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14
Q

How do you transfect insect cells with the gene?

A

by a virus

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15
Q

What are the advantages of mammalian recombinant protein expression?

A
  • correct fold
  • recreated into the cytosol
  • no weight limit
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16
Q

What are the disadvantages of mammalian recombinant protein expression?

A
  • low yields
  • fragile cells
  • infectious impurities
  • complicated media
17
Q

what are the advantages of mammalian transgenic protein expression?

A
  • high yield
  • secreted ( via milk for example)
  • correct fold
18
Q

what are the disadvantages of mammalian transgenic protein expression?

A
  • canbe toxic to the animal
  • fragile cells
  • infectious impurities
  • high cost
19
Q

How do you homogenise mammalian tissue?

A
  • cut up into small pieces, then blender, high salt concentration and keep cool
20
Q

How do you homogenise fragile plant tissues?

A

blend then grind

21
Q

How do you homogenise fibrous plant sources?

A

grind with sand or place in liquid nitrogen

22
Q

What is the point of homogenisation?

A

break open tissues to get the cells out

23
Q

What are the two methods to lyse cells?

A
  • physical

- non - mechanical methods

24
Q

Examples of physical mechanical cell disruption

A
  • blender
  • Grind in nitrogen
  • grind with acid washed sand
  • compression and expansion
  • ultrasonicator
25
Q

Examples of liquid shear physical cell disruption

A
  • French press homogeniser
  • suspension forced through narrow space
  • only small volumes , difficult to clean
26
Q

What is sonication?

A
  • physical disruption of cells
27
Q

How does sonication work?

A

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

28
Q

What are the drawback of mechanical methods?

A
  • heat (denaturation)

- hearing ( need sonic cabinets)

29
Q

What are the 4 non - mechanical lysis techniques?

A
  • osmotic lysis
  • freeze
  • detergent solubilisation
  • lytic enzyme damage cell walls
30
Q

What is subcellular fractionation?

A

Cell fractionation is the process used to separate cellular components while preserving individual functions of each component.

31
Q

What are the 4 steps for protein extraction?

A
  • Homogenisation
  • Cell lysis
  • Subcellular fractionation
  • purification
32
Q

What is a suitable cell lysis method for animal tissue?

A

Blender

33
Q

What is a suitable cell lysis method for animal cell culture?

A

physical

  • blender
  • compression/expansion

Non - mechanical

  • osmotic shock
  • freeze thaw
  • lytic enzyme
34
Q

What is a suitable cell lysis method for plant tissue?

A

Physical

  • Blender
  • Grind in liquid N2
  • Grind with acid washed sand
35
Q

What is a suitable cell lysis method for plant cell culture?

A

Physical

  • Blender
  • Grind in liquid N2
  • Grind with acid washed sand
  • compression/ expansion
  • ultrasonicator

Non - mechanical
- Freeze

36
Q

what is a suitable cell lysis method for bacteria?

A

Physical

  • -Grind in liquid N2
  • Grind with acid washed sand
  • compression/ expansion
  • ultrasonicator

Non mechanical

  • Freeze
  • detergent
37
Q

what is a suitable cell lysis method for yeast?

A
  • grind in liquid N2
  • ultrasonicator
  • Freeze
  • enzyme
38
Q

What are drawbacks of mechanical methods?

A
  • heat
  • denaturation
  • hearing

BB10186: Proteins purification and characterisation (10 decks)

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