[a fresh student]

I am solving past exam papers and can’t tackle this question. If anybody could help with any of the questions…

Question A 1. (Compulsory) [20 Marks]

A biochemist wishes to purify from leaves of the spinach plant the soluble electron transfer protein with the following amino acid sequence:
VEVLWGGDDGSLAFLPGDFSVASGEEIVFKNN AGFPHNVVFDEDEIPSGVDAAKISMSEEDLLN APGETYKVTLTEKGTYRFYCSPHQGAGMVGKV TVN

Each molecule of the protein, which is a monomer and has no enzyme activity, binds strongly to a single atom of copper that has two oxidation states. The reduced Cu(I) form can be readily prepared by addition of the small molekule ascorbate and is colourless. The oxidised Cu(II) form, which can be prepared by addition of the inorganic ion ferricyanide, is a deep blue colour with an absorption maximum of 597 nm, in addition to the normal absorption band at 280nm. The three-dimensional structure and other macroscopic physicochemical properties of the protein are otherwise unaltered by the change in oxidation state.

a. Suggest a sensible method for purification of the protein. Give as much
detail as you can. [12 Marks]
b. How might the optical absorbance of the protein be used to monitor the
purity of the protein? [2 Marks]
c. How might you determine the secondary structure content of the purified
protein? [2 Marks]
d. A molecular biologist clones the gene that encodes this protein into a
bacterial vector that can be used to over-express the protein in a
hexahistidine- (His6-) tagged form that is produced in inclusion bodies.
Indicate how you would modify the purification strategy to deal with the
inclusion bodies and obtain the purified protein. [2 Marks]
e. Given the chance to repeat the experiment in Part (d) how might you avoid
the production of inclusion bodies. [2 Marks]

[Coursework.info]

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[gumball]

Seeing as you have no answers, I might as well try and chip in and give you some points to look into and [quite probably] dismiss as wrong. I haven't done much [wet] experimental work on proteins, but rather work on theoretical stuff, thus avoiding all this.

a) Run a gel. Various bits on here: http://en.wikipedia.org/wiki/Protein_purification . This will separate the proteins into bands based on various chemical properties (not to mention size).

b) Unsure. Shining UV light onto it will illuminate the aromatic residues. Maybe this is completely unrelated, but.. hey, it's something! http://www.biotek.com/resources/arti...orescence.html

c) Circular dichroism would work. http://en.wikipedia.org/wiki/Circular_dichroism

d) e) Pass.

[onefour]

To add to the above post:
a. Not sure how much detail to put here but it involves a lot of centrifuging, chromatography and running gels
b. Possibly Bradford assay - add some Biorad reagent, make a standard curve, calculate protein concentration of samples taken from each purification procedure
d. This assumes you put something about chromatography in part A so you could use Immobilised Metal Ion Affinity Chromatography (IMAC) - the imidazole rings in the histidine co-ordinate with metal ions in the column so the protein of interest is retained and washed out with a buffer containing a high concentration of imidazole that competes for the metal ions
e. I don't know whether this answer is right in the context of the question or not but there are several ways I know of to reduce inclusion bodies - low affinity promoters fused to the gene of interest, culturing the bacteria at a lower temperature and, very rarely, co-expressing molecular chaperones