Lecture 7 Flashcards
Purification and assays of recombinant proteins
What 3 properties of proteins are tradition method to purify them based on?
size
charge
hydrophobocicity
How pure does your protein have to be?
never more than is required by end user - quite gavalar.
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
What are the 2 main fusion proteins used to purify proteins
glutathionine S transferase
maltose binding protein
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
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
For the MBP method what is required to export the protein to the periplasm and increased solubility?
it must be a N-terminal fusion
Are MBP and GST tags large or small?
LARGE like these letters.
What is the problem with large fusion proteins
may modify properties of recomb prot
How can the problem caused by large fusion proteins be overcome?
Remove them by including specific protease cleavage site between recomb and fusion protein
What does TAP tagging stand for
Tandem affinity purification tagging
What does TAP tagging allow?
purification of recombinant proteins expressed at LOW levels in cell
What are the 3 segments of the TAP tag
starting from C terminus: Calmodulin binding peptide, TEV protease cleavage site, Protein A.
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
What is this a particularly useful method for
purifying native complexes of proteins within the cell
What is the method used to quantify protein purification
Ultraviolet absorption protein assay
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
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)
How are recomb proteins monitored through visualising
detection of protein bands in electrophoresis gells
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
what is the pI
pH at which protein has no net charge
what does the charge proteins carry depend on
pH of solution
What would you ideally use to monitor purification
ENZYMATIC assay specific to protein you’re purifying
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)
