Lecture 1

Question 1

What are the reasons we isolate a protein?

Answer 1

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

Question 2

How pure do we need proteins?

Answer 2

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

Question 3

What are the two protein sources?

Answer 3

natural vs recombinant

Question 4

What are two requirements of obtaining proteins from natural sources?

Answer 4

• must be adundant and stable

- source must be readily available

Question 5

What are the disadvantages of plant proteins sources?

Answer 5

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

Question 6

What is the advantage of natural sourced proteins?

Answer 6

in vivo

Question 7

What are examples of recombinant protein sources?

Answer 7

• bacteria ,yeast , insect , plant cells

- transgenic animals

Question 8

What is an advantage of recombinant sources?

Answer 8

yields a lot higher

Question 9

What are advantages of prokaryotic protein expression?

Answer 9

• rapid growth

- simple nutrition

Question 10

What are disadvantages of prokaryotic protein expression?

Answer 10

• no post- translation modification

- often insoluble

Question 11

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

Answer 11

• good growth
• simple to grow
• genetics understood well

Question 12

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

Answer 12

• misfolding
  hyperglycosylation
  -not high cell density

Question 13

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

Answer 13

pros

• higher cell density
• grows on methanol

cons
- still misfolds

Question 14

How do you transfect insect cells with the gene?

Answer 14

by a virus

Question 15

What are the advantages of mammalian recombinant protein expression?

Answer 15

• correct fold
• recreated into the cytosol
• no weight limit

Question 16

What are the disadvantages of mammalian recombinant protein expression?

Answer 16

• low yields
• fragile cells
• infectious impurities
• complicated media

Question 17

what are the advantages of mammalian transgenic protein expression?

Answer 17

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

Question 18

what are the disadvantages of mammalian transgenic protein expression?

Answer 18

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

Question 19

How do you homogenise mammalian tissue?

Answer 19

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

Question 20

How do you homogenise fragile plant tissues?

Answer 20

blend then grind

Question 21

How do you homogenise fibrous plant sources?

Answer 21

grind with sand or place in liquid nitrogen

Question 22

What is the point of homogenisation?

Answer 22

break open tissues to get the cells out

Question 23

What are the two methods to lyse cells?

Answer 23

• physical

- non - mechanical methods

Question 24

Examples of physical mechanical cell disruption

Answer 24

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

Question 25

Examples of liquid shear physical cell disruption

Answer 25

- French press homogeniser - suspension forced through narrow space - only small volumes , difficult to clean

Question 26

What is sonication?

Answer 26

- physical disruption of cells

Question 27

How does sonication work?

Answer 27

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

Question 28

What are the drawback of mechanical methods?

Answer 28

- heat (denaturation) | - hearing ( need sonic cabinets)

Question 29

What are the 4 non - mechanical lysis techniques?

Answer 29

- osmotic lysis - freeze - detergent solubilisation - lytic enzyme damage cell walls

Question 30

What is subcellular fractionation?

Answer 30

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

Question 31

What are the 4 steps for protein extraction?

Answer 31

- Homogenisation - Cell lysis - Subcellular fractionation - purification

Question 32

What is a suitable cell lysis method for animal tissue?

Answer 32

Blender

Question 33

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

Answer 33

physical - blender - compression/expansion Non - mechanical - osmotic shock - freeze thaw - lytic enzyme

Question 34

What is a suitable cell lysis method for plant tissue?

Answer 34

Physical - Blender - Grind in liquid N2 - Grind with acid washed sand

Question 35

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

Answer 35

Physical - Blender - Grind in liquid N2 - Grind with acid washed sand - compression/ expansion - ultrasonicator Non - mechanical - Freeze

Question 36

what is a suitable cell lysis method for bacteria?

Answer 36

Physical - -Grind in liquid N2 - Grind with acid washed sand - compression/ expansion - ultrasonicator Non mechanical - Freeze - detergent

Question 37

what is a suitable cell lysis method for yeast?

Answer 37

- grind in liquid N2 - ultrasonicator - Freeze - enzyme

Question 38

What are drawbacks of mechanical methods?

Answer 38

- heat - denaturation - hearing