Lecture 7

Question 1

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

Answer 1

size
charge
hydrophobocicity

Question 2

How pure does your protein have to be?

Answer 2

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

Question 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

Answer 3

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

Question 4

What are the 2 main fusion proteins used to purify proteins

Answer 4

glutathionine S transferase

maltose binding protein

Question 5

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

Answer 5

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

Question 6

What is structure of MBP and how does it work

Answer 6

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

Question 7

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

Answer 7

it must be a N-terminal fusion

Question 8

Are MBP and GST tags large or small?

Answer 8

LARGE like these letters.

Question 9

What is the problem with large fusion proteins

Answer 9

may modify properties of recomb prot

Question 10

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

Answer 10

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

Question 11

What does TAP tagging stand for

Answer 11

Tandem affinity purification tagging

Question 12

What does TAP tagging allow?

Answer 12

purification of recombinant proteins expressed at LOW levels in cell

Question 13

What are the 3 segments of the TAP tag

Answer 13

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

Question 14

Describe how TAP tagging works to purify recomb protein

Answer 14

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

Question 15

What is this a particularly useful method for

Answer 15

purifying native complexes of proteins within the cell

Question 16

What is the method used to quantify protein purification

Answer 16

Ultraviolet absorption protein assay

Question 17

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

Answer 17

A280

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

Question 18

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

Answer 18

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

Question 19

How are recomb proteins monitored through visualising

Answer 19

detection of protein bands in electrophoresis gells

Question 20

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

Answer 20

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

Question 21

what is the pI

Answer 21

pH at which protein has no net charge

Question 22

what does the charge proteins carry depend on

Answer 22

pH of solution

Question 23

What would you ideally use to monitor purification

Answer 23

ENZYMATIC assay specific to protein you’re purifying

Question 24

Give an example of an enzymatic assay

Answer 24

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)

Question 25

What do you usually add the protein preparation to?

Answer 25

buffered solution of substrate at standardised temp and pH

Question 26

Generally how do you go about performing and enzymatic assay

Answer 26

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

Question 27

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

Answer 27

extinction coefficient
fluoresence
pH

Question 28

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

Answer 28

somehow label ligand so ligand binding can be ,measured

Question 29

Describe a new assay for DNAsel

Answer 29

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