LECTURE 1 (PROTEIN PURE) Flashcards Preview

BB10186 > LECTURE 1 (PROTEIN PURE) > Flashcards

Flashcards in LECTURE 1 (PROTEIN PURE) Deck (69)
Loading flashcards...
1

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

3D structure maintained

2

Proteins hydrophobic residues are localised mostly where?

Core, although there are hydrophobic patches

3

Purification of protein is all about ?

exploiting its individual properties

4

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

Its purified

5

Purification percentage aim (moderate)

95%

6

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

unexpected side effects

7

Low cost

high volume

8

High cost

low volume

9

examples of low cost protein production

industrial bulk enzyme

10

Antigen for antibody sequencing purification percentage ?

greater than 95%

11

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

high (greater than 95)

12

Therapeutic protein use

greater than 99.9%

13

Aim of protein purification?

Aim: High yield retaining maximum activity

14

four steps of protein purification?

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

15

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

- Abundant
- Stable
- Readily available
- Wide range

16

Advantages of protein purification?

Formed in vivo

17

Disadvantages of protein purification- natural sources ?

Abundance and reproducibility
-

18

Plant sources often _______

low in abundance (proteins)
- oxidation occurring
- seeds in large quantities to get a large abundance
- Fruits undergo developmental changes (particular ripeness at particular times)

19

Recombinant sources for protein preparation?

Clone gene into a plasmid, overexpress
- Usually bacteria (e.coli)
- or transgenic animals

20

Yield of recombinant sources of protein production

yields may be 100-fold higher

21

When is recombinant sources of protein production most successful ?

In similar organism to which it originated from

22

how is over expression in pro's achieved?

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

23

Advantages of Prokaryotic expression?

Rapid growth, simple nutrition

24

disadvantages of Prokaryotic expression?

No post- translational modifications
often insoluble

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