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BB10186 > LECTURE 1 (PROTEIN PURE) > Flashcards

LECTURE 1 (PROTEIN PURE) Flashcards

1
Q

When we purify proteins we want to keep them in what kind of formation?

A

3D structure maintained

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2
Q

Proteins hydrophobic residues are localised mostly where?

A

Core, although there are hydrophobic patches

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3
Q

Purification of protein is all about ?

A

exploiting its individual properties

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4
Q

Until what point, can we look at the kinetics of an enzyme?

A

Its purified

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5
Q

Purification percentage aim (moderate)

A

95%

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6
Q

If a protein isn’t pure it can cause ? (drugs)

A

unexpected side effects

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7
Q

Low cost

A

high volume

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8
Q

High cost

A

low volume

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9
Q

examples of low cost protein production

A

industrial bulk enzyme

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10
Q

Antigen for antibody sequencing purification percentage ?

A

greater than 95%

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11
Q

If STRUCTURE and CHARACTERISATION is being explored of a protein, purification percentage must be ??

A

high (greater than 95)

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12
Q

Therapeutic protein use

A

greater than 99.9%

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13
Q

Aim of protein purification?

A

Aim: High yield retaining maximum activity

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14
Q

four steps of protein purification?

A

1) Preparation extraction and clarification
2) Capture
3) Intermediate purification
4) Polishing

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15
Q

In order to purify a protein its properties must consist of which four things ?

A
  • Abundant
  • Stable
  • Readily available
  • Wide range
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16
Q

Advantages of protein purification?

A

Formed in vivo

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17
Q

Disadvantages of protein purification- natural sources ?

A

Abundance and reproducibility

-

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18
Q

Plant sources often _______

A

low in abundance (proteins)

  • oxidation occurring
  • seeds in large quantities to get a large abundance
  • Fruits undergo developmental changes (particular ripeness at particular times)
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19
Q

Recombinant sources for protein preparation?

A

Clone gene into a plasmid, overexpress

  • Usually bacteria (e.coli)
  • or transgenic animals
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20
Q

Yield of recombinant sources of protein production

A

yields may be 100-fold higher

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21
Q

When is recombinant sources of protein production most successful ?

A

In similar organism to which it originated from

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22
Q

how is over expression in pro’s achieved?

A

Genes for protein of interest are under the control of a strong promotor

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23
Q

Advantages of Prokaryotic expression?

A

Rapid growth, simple nutrition

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24
Q

disadvantages of Prokaryotic expression?

A

No post- translational modifications

often insoluble

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25
Q

How to overcome insolubility of proteins?

A

Add detergent and refold the protein (not always successful)

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26
Q

Eukaryotic expression advantages?- YEAST -(S. cerevisiae)

A
  • good growth rates
  • Simple (cheap) media
  • Genetics understood
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27
Q

Eukaryotic expression disadvantages?- YEAST (S. cerevisiae)

A

Misfolding

hyperglycosylation (adding too many glucose groups)

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28
Q

Pichia pastoris advantages vs disadvantages (eu expression)

A

higher cell density, grows on methanol, still misfolds

29
Q

Insect cells driven expression by??

A

infect cells with baculovirus (which contains the gene of interest)»» protein production

30
Q

Advantages of mammalian recombinant expression?

A

Correct fold
secreted
no MW limit

31
Q

diadvantages of mammalian recombinant expression?

A

Low yield (mg/litre)
Fragile cells
Infectious impurities
Complicated media

32
Q

disadvantages of mammalian transgenic expression (milk)

A

Toxicity (high concentration)
Fragile cells
Infectious impurities
Cost

33
Q

advantages of mammalian transgenic expression (milk)

A

Yield (>10g/litre)
Secreted (milk)
Correct fold

34
Q

homogenisation

A

Break open the tissues to get the cells out

35
Q

Method of homogenisation mammalian tissue

A

Cut into smaller pieces and then blend (isotonic buffer)

36
Q

Ease of homogenisation is dependent on?? link to an example?

A

Tissue connectivity
eg lung- harder
brain- easier

37
Q

Plant tissue homogenisation

A

Blender (soft)

grinding with pestle and mortar for fibrous tissues first (stems)

38
Q

When might we freeze in N2 ??

A

Homogenisation

  • to make the protein more fragile
  • protein needs to be stable at minus 90
39
Q

Cell lysis

A

Breaking of plasma membrane (cell death)

40
Q

Cell lysis is easier for and why?

A

Easiest for eukaryotic cells

due to the absence of the cell wall

41
Q

two general names of the methods used for cell lysis?

A

Physical (mechanical and liquid shear) or non-mechanical methods

42
Q

Bacteria cell lysis difficulty??

A

Peptigylcan which is difficult to break down (cell wall monomers)

43
Q

Mechanical methods of cell disruption?

A

rotating blades e.g. Waring blender
grinding in pestle & mortar
vortexing with glass beads

44
Q

Liquid shear methods for cell disruption

A

e.g. French Press homogenizer
suspension forced through narrow space
Pressure release bursts cells

45
Q

Issues with French press homogeniser?

A

only small volumes, difficult to clean therefore only used in labs

46
Q

Blenders have??

A

stainless steal tops to ensure cooling materisal (reduce activity of the proteases)

47
Q

Sonnication method of physical cell disruption

A

High frequency sound waves

  • ossillating metal probs
  • shear cells
  • HEAT AND PRESSURE
  • pressure»> cell burs and contents released
48
Q

Drawbacks of mechanical methods and how its overcame

A 
heat 
(pauses, ice)
denaturation 
(less vigorous)
hearing 
(sonic cabinets)
49
Q

Non-mechanical lysis, 4 methods?

A
  • osmotic lysis
  • freeze/thaw
  • detergent solubilisation e.g. BugBuster®
  • Lytic enzymes damage cell walls
50
Q

Osmotic lysis relies on ?

A

Hypertonic solution (water potential of 0)

51
Q

Osmotic lysis can be used on?

A

Insect and mammalian cells

52
Q

Freeze/thaw lysis works by?

A

Ice crystals forming and expanding in cells and causing them to burst open

53
Q

detergent solubilisation e.g. BugBuster®

works by?

A

Non-ionic denaturing of cells

54
Q

Lytic enzyme work by?

A

Damaging cell walls

55
Q

Lytic enzymes use is followed by?

A

Osmotic shock

56
Q

examples of lytic enzymes??

A

Application: lipases and proteases

57
Q

Cell types for mechanical and non mechanical cell lysis?

A

mechanical- plant, microbe

non-mechanical- animal

58
Q

Advantages of mechanical methods of cell lysis?

A

Can overcome tough cell walls

59
Q

disadvantages of mechanical methods of cell lysis?

A

Heat, Damage, Denaturation of the proteins (depending on its opt)

60
Q

advantages of non-mechanical methods of cell lysis?

A

Mild, selective

61
Q

Disadvantages of non-mechanical methods of cell lysis?

A

Expensive, extra purification needed, cell/product specific

62
Q

Mechanical and non mechanical methods may be?

A

combine for a particular purpose

63
Q

How do we persevere protein activity in cell lysis?

A

buffer, salts, EDTA, protease inhibitors, detergents, etc.- NEEDS TO BE OPTIMISED
Chill to 4°C (stop protease activity)

64
Q

Supernatant?

A

soluble fraction

65
Q

Pellet

A

insoluble fraction

66
Q

Low speed centrifugation?

A

1000g for 10 mins

67
Q

medium speed centrifugation?

A

20 000g 20 mins

68
Q

high speed centrifugation?

A

80 000g 1 hour

69
Q

very high speed centrifugation?

A

150 000g 3 hours

BB10186 (6 decks)

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