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BB10186 > Lec 1 Crennell > Flashcards

Lec 1 Crennell Flashcards

1
Q

What are the 4 main protein properties?

A
  • polypeptide chain
  • folded state as purified
  • held together by non covalent interactions
  • have charge, hydrophobicity, affinity
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2
Q

Why purify a protein?

A
  • 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
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3
Q

How pure is pure?

A

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

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4
Q

Proteins can be naturally sourced. What are the requirements of proteins which are gained in this way, and some of their features?

A
  • 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
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5
Q

What must you ensure when gaining a protein from transgenics and in which organisms is it most successful?

A
  • must make sure native protein is knocked out though otherwise will have difficulty separating the two
  • most successful in similar organisms
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6
Q

Breif overview of prokaryotic expression of protein, how does it work?

A
  • gene in plasmid under strong promotor control

- expression is induced

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7
Q

Advantages of Prokaryotic expression of protein

A
  • rapid growth

- simple nutrition

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8
Q

Disadvantages of Prokaryotic expression of protein

A
  • No post-translational modification

- Often insoluble

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9
Q

Issues with using prokaryotes for protein expression

A
  • post translational modifications wont happen in bacteria that happen in eukaryotes, e.g. sugars not added etc
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10
Q

Eukaryotic protein expression (Yeast) advantages

A
  • Good growth rates
  • simple
  • cheap
  • genetics understood
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11
Q

Eukaryotic protein expression (Yeast) disadvantages

A
  • misfolding
  • hyperglycosylation
  • not high cell density
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12
Q

Mammalian recombinant expression advantages

A
  • correct fold
  • secreted
  • no molecular weight limit
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13
Q

Mammalian recombinant expression disadvantages

A
  • low yield
  • expensive
  • 2 months
  • fragile cells
  • infections impurities
  • complicated media
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14
Q

Mammalian transgenic expression (goat milk etc) advantages

A
  • yield relatively good
  • secreted in milk
  • correct fold
  • only expressed in milk
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15
Q

Mammalian transgenic expression (goat milk etc) disadvantages

A
  • must make sure what you’re expressing isn’t toxic
  • fragile cells
  • infectious impurities
  • cost is high
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16
Q

Why is it better to have organisms close together in expression?

A
  • will express better but it is expensive in some cases so if yo can use bacteria then do
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17
Q

How does extraction of protein homogenisation (natural sources)
in MAMMALIAN work?

A

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

18
Q

How does extraction of protein homogenisation (natural sources)
in PLANTS work?

A
  • 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)
19
Q

Cell lysis used to extraction proteins. What is cell lysis?

A
  • breaking of plasma membrane (cell death)
20
Q

Why must the release of proteases in extraction of protein Cell lysis be stopped?

A

proteases will break down everything even your desired protein

21
Q

What is the order of ease in Extraction of protein via Cell lysis?

A
  • Eukaryotic easiest
  • bacteria tricky as peptidoglycan wall
  • Plants hardest as heavy cellulose wall
22
Q

Extraction of protein Cell lysis - PHYSCIAL CELL DISRUPTION

MECHANICAL, how does it work?

A
  • Rotating blades e.g. waring blender
  • grinding in pestle and mortar (with acid sand or N2)
  • vortexing with glass beads
23
Q

Extraction of protein Cell lysis - PHYSCIAL CELL DISRUPTION

LIQUID SHEAR, how does it work?

A
  • French press homogenizer
  • suspension forced through narrow space (pressure release bursts cells)
  • only small volumes/difficult to clean
24
Q

Extraction of protein Cell lysis - PHYSCIAL CELL DISRUPTION

SONICATION, how does it work?

A
  • 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)
25
Q

Draw backs of physcial methods

A
  • Heat used: can denature proteins. Overcome this by putting pauses in and keeping on ice doing bursts of heat at a time.
  • hearing can be affected in sonic cabinets
26
Q

Extraction of protein Cell lysis - NON MECHANICAL

Osmotic Lysis, how does it work?

A
  • place insect/mammalian cells in hypotonic solution
27
Q

Extraction of protein Cell lysis - NON MECHANICAL

Freeze/thaw, how does it work?

A
  • water expands on freezing so can break open cells
28
Q

Extraction of protein Cell lysis - NON MECHANICAL

Detergent solubilisation, how does it work?

A
  • detergents are non ionic so they don’t denature proteins
  • can be affective
  • e.g. BugBuster
29
Q

Extraction of protein Cell lysis - NON MECHANICAL

Lytic enzymes, how does it work?

A
  • damage cells walls
  • then do osmotic shock lysis to break open membrane
  • depends on types of cells e.g. lipases, proteases
30
Q

Mechanical advantages

A

Tough cell walls can be broken

31
Q

Non mechanical disadvantages

A
  • expensive, extra purification needed, cell product specific
32
Q

Types of cells which can use Non mechanical lysis

A

animal cell suspension

33
Q

Types of cells which can use mechanical lysis

A

plant and microbe

34
Q

Physical advantages

A

tough cell walls can be broken still

35
Q

Mechanical disadvantages

A
  • heat damage and denaturation
36
Q

What do you need to ensure when combining both types of cell lysis?

A
  • need to preserve activity
  • Medium - buffer, salts, EDTA, protease inhibitors, detergents etc
  • need optimisation
  • chill to 4 degrees C
37
Q

Extraction of proteins - what is SUBCELLULAR FRACTIONATION?

A
  • for eukaryotic cells
  • gentle disruption so retains compartments
  • uses centrifugation (extracts based on size)
38
Q

Ads and Disads of naturally sourced proteins

A
  • advantage is that you can form in vivo

- disadvantage: abundance, reproducibility

39
Q

Which type of cell is cell lysis most easiest for and what is released when it occurs in this cell?

A
  • easiest for eukaryotes, releases cell contents, proteins, proteases and nucleic acids
40
Q

What are the two types of cell lysis?

A
  • physical or non-mechanical

BB10186 (4 decks)

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