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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
How to overcome insolubility of proteins?
Add detergent and refold the protein (not always successful)
26
Eukaryotic expression advantages?- YEAST -(S. cerevisiae)
- good growth rates - Simple (cheap) media - Genetics understood
27
Eukaryotic expression disadvantages?- YEAST (S. cerevisiae)
Misfolding | hyperglycosylation (adding too many glucose groups)
28
Pichia pastoris advantages vs disadvantages (eu expression)
higher cell density, grows on methanol, still misfolds
29
Insect cells driven expression by??
infect cells with baculovirus (which contains the gene of interest)>>>> protein production
30
Advantages of mammalian recombinant expression?
Correct fold secreted no MW limit
31
diadvantages of mammalian recombinant expression?
Low yield (mg/litre) Fragile cells Infectious impurities Complicated media
32
disadvantages of mammalian transgenic expression (milk)
Toxicity (high concentration) Fragile cells Infectious impurities Cost
33
advantages of mammalian transgenic expression (milk)
Yield (>10g/litre) Secreted (milk) Correct fold
34
homogenisation
Break open the tissues to get the cells out
35
Method of homogenisation mammalian tissue
Cut into smaller pieces and then blend (isotonic buffer)
36
Ease of homogenisation is dependent on?? link to an example?
Tissue connectivity eg lung- harder brain- easier
37
Plant tissue homogenisation
Blender (soft) | grinding with pestle and mortar for fibrous tissues first (stems)
38
When might we freeze in N2 ??
Homogenisation - to make the protein more fragile - protein needs to be stable at minus 90
39
Cell lysis
Breaking of plasma membrane (cell death)
40
Cell lysis is easier for and why?
Easiest for eukaryotic cells | due to the absence of the cell wall
41
two general names of the methods used for cell lysis?
Physical (mechanical and liquid shear) or non-mechanical methods
42
Bacteria cell lysis difficulty??
Peptigylcan which is difficult to break down (cell wall monomers)
43
Mechanical methods of cell disruption?
rotating blades e.g. Waring blender grinding in pestle & mortar vortexing with glass beads
44
Liquid shear methods for cell disruption
e.g. French Press homogenizer suspension forced through narrow space Pressure release bursts cells
45
Issues with French press homogeniser?
only small volumes, difficult to clean therefore only used in labs
46
Blenders have??
stainless steal tops to ensure cooling materisal (reduce activity of the proteases)
47
Sonnication method of physical cell disruption
High frequency sound waves - ossillating metal probs - shear cells - HEAT AND PRESSURE - pressure>>> cell burs and contents released
48
Drawbacks of mechanical methods and how its overcame
``` heat (pauses, ice) denaturation (less vigorous) hearing (sonic cabinets) ```
49
Non-mechanical lysis, 4 methods?
- osmotic lysis - freeze/thaw - detergent solubilisation e.g. BugBuster® - Lytic enzymes damage cell walls
50
Osmotic lysis relies on ?
Hypertonic solution (water potential of 0)
51
Osmotic lysis can be used on?
Insect and mammalian cells
52
Freeze/thaw lysis works by?
Ice crystals forming and expanding in cells and causing them to burst open
53
detergent solubilisation e.g. BugBuster® | works by?
Non-ionic denaturing of cells
54
Lytic enzyme work by?
Damaging cell walls
55
Lytic enzymes use is followed by?
Osmotic shock
56
examples of lytic enzymes??
Application: lipases and proteases
57
Cell types for mechanical and non mechanical cell lysis?
mechanical- plant, microbe non-mechanical- animal
58
Advantages of mechanical methods of cell lysis?
Can overcome tough cell walls
59
disadvantages of mechanical methods of cell lysis?
Heat, Damage, Denaturation of the proteins (depending on its opt)
60
advantages of non-mechanical methods of cell lysis?
Mild, selective
61
Disadvantages of non-mechanical methods of cell lysis?
Expensive, extra purification needed, cell/product specific
62
Mechanical and non mechanical methods may be?
combine for a particular purpose
63
How do we persevere protein activity in cell lysis?
buffer, salts, EDTA, protease inhibitors, detergents, etc.- NEEDS TO BE OPTIMISED Chill to 4°C (stop protease activity)
64
Supernatant?
soluble fraction
65
Pellet
insoluble fraction
66
Low speed centrifugation?
1000g for 10 mins
67
medium speed centrifugation?
20 000g 20 mins
68
high speed centrifugation?
80 000g 1 hour
69
very high speed centrifugation?
150 000g 3 hours

BB10186 (6 decks)

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