Lecture 7 Flashcards Preview

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Flashcards in Lecture 7 Deck (29):
1

What 3 properties of proteins are tradition method to purify them based on?

size
charge
hydrophobocicity

2

How pure does your protein have to be?

never more than is required by end user - quite gavalar.

3

For each type of product, how pure should they be.
Product types are: bulk enzyme
therapeutic drugs
basic science usage
overexpressed proteins to look for new enzymes
proteins for in vitro ligand binding/structural studies

bulk impurities removed
>99.9% pure.
variable
use a crude lyase
>99% pure

4

What are the 2 main fusion proteins used to purify proteins

glutathionine S transferase
maltose binding protein

5

What is the structure of a GST and how does it work

dimeric 26kDa, binds with high affinity to glutathione. They are a family of enzymes involved in cellular defenses against electrophilic stress
glutathione attached to column via agarose beads
Fusion protein binds to these
Then elute protein with high conc of free glutathione, to compete for binding sites

6

What is structure of MBP and how does it work

periplasmic protein, part of ABC transporter
Recognises maltose and longer maltodextrins with high affinity
Also binds to amylose with high affinity
Cross link amylose to solid matrix on colum
MBP fusion protein binds to the amylose
Elute with excess of maltose

7

For the MBP method what is required to export the protein to the periplasm and increased solubility?

it must be a N-terminal fusion

8

Are MBP and GST tags large or small?

LARGE like these letters.

9

What is the problem with large fusion proteins

may modify properties of recomb prot

10

How can the problem caused by large fusion proteins be overcome?

Remove them by including specific protease cleavage site between recomb and fusion protein

11

What does TAP tagging stand for

Tandem affinity purification tagging

12

What does TAP tagging allow?

purification of recombinant proteins expressed at LOW levels in cell

13

What are the 3 segments of the TAP tag

starting from C terminus: Calmodulin binding peptide, TEV protease cleavage site, Protein A.

14

Describe how TAP tagging works to purify recomb protein

protein A binds to IGg on first column.
Eluted off using protease
Wash elute onto second column containing calmodium and Ca2+
Elute with EGTA

15

What is this a particularly useful method for

purifying native complexes of proteins within the cell

16

What is the method used to quantify protein purification

Ultraviolet absorption protein assay

17

In the UAPA at what wavelength will proteins absorb light and why?

A280
cos of aromatic residues on protein and a small contribution from cystine groups

18

How is the theoretical molecular extinction coefficient for a protein of known sequence calculated

no. of Trpxno. of tyrxno. of disulphide binds = epsilon280 (M-1cm-1)

19

How are recomb proteins monitored through visualising

detection of protein bands in electrophoresis gells

20

Give 3 examples of specific methods used to visualise proteins and details

Coomassie Brilliant Blue
dye binds to +vely charged residues (Arg/lys/his)
quick
detect 1ug per band

Silver stain
More laborious
0.1ug per band
based on reduction of ionic silver nitrate to metallic silver by protein already in gel

Isoelectric focusing
Seperate proteins using native electrophoresis on gel containing pH gradient.
Protein goes to it's pI
Use in combination with SDS-phage in 2d electrophoresis

21

what is the pI

pH at which protein has no net charge

22

what does the charge proteins carry depend on

pH of solution

23

What would you ideally use to monitor purification

ENZYMATIC assay specific to protein you're purifying

24

Give an example of an enzymatic assay

DNase activity of sample of rhDNAsel protein can be assessed to access the specific activity
(mol of substrate used/product formed per unit time per mg protein)

25

What do you usually add the protein preparation to?

buffered solution of substrate at standardised temp and pH

26

Generally how do you go about performing and enzymatic assay

once in standardised buffer, measure initial rate of substrate use/product formation
by following a parameter

27

What sorts of parameters can be measured to measure protein presence in enzymatic assay

extinction coefficient
fluoresence
pH

28

What do you do if protein binds to a particular molecule, and you need to do enzymatic assay?

somehow label ligand so ligand binding can be ,measured

29

Describe a new assay for DNAsel

use dsDNA stained with fluorescent dye. When enzyme digests dna , dye can't bind and fluorescence drops.