What fractionation procedure could be used to purify protein 1 from a mixture of three proteins whose amino acid compositions are as follows?

1. 25% Ala, 20% Gly, 20% Ser, 10% Ile, 10% Val, 5% Asn, 5% Gln, 5% Pro

2. 30% Gln, 25% Glu, 20% Lys, 15% Ser, 10% Cys

3. 25% Asn, 20% Gly, 20% Asp, 20% Ser, 10% Lys, 5% Tyr

All three proteins are similar in size and pI, and there is no antibody available for protein 1.

Short Answer

Expert verified

Ion Exchange Chromatography can be used to purify protein 1 from the mixture of given three proteins.

Step by step solution

01

Ion Exchange Chromatography

Ion Exchange Chromatography is a fractionation process used to separate charged molecules (anions and cations). In this process, charged molecules attach to oppositely charged groups which are chemically bound to a matrix such as cellulose or agarose.

On anion exchangers, anions bind to cationic groups, while on cation exchangers, cations bind to anionic groups. A matrix with linked diethylaminomethyl (DEAE) groups is perhaps the most commonly used anion exchanger, whereas a matrix with attached carboxymethyl (CM) groups is probably the most commonly used cation exchanger.

02

Process of Ion Exchange Chromatography

The proteins to be extracted are dissolved in a buffer with proper pH and salt concentration before being transferred to an ion exchanger column. The buffer is then used to wash the column. Proteins with low affinities for the ion exchanger pass through the column faster than proteins with higher affinities as the column is washed. The column effluent is then collected in a number of fractions. Proteins that bind tightly to the column's ion exchanger can be eluted (washed through the column) by using an eluant (a buffer with a greater salt concentration or a pH that lowers the matrix's affinity for the protein).

Fig: Purification by Ion Exchange Chromatography

03

Explanation

When the mixture containing the 3 proteins is passed through an ion exchanger column containing an anion exchanger, protein 2 and protein 3 will bind to these anion exchangers. Protein 2 and protein 3 contain aspartic acid and glutamic acid which are negatively charged amino acids. Protein 1 will pass through the column at a faster rate and will be eluted out of the column. Protein 2 and protein 3 can be eluted by using an eluant. In this way we can separate protein 1 from the mixture of given 3 proteins.

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Most popular questions from this chapter

Question: Sketch a phylogenetic tree for the family of homologous proteins

whose partial sequences are given below.

Protein A T L A D K A I S L H D S

Protein B T L G D K A V S I H E S

Protein C T L A D K A I S V H D S

Consult Table 5-1 to complete the following: (a) On a plot of absorbance at 280 nm versus elution volume, sketch the results of gel filtration of a mixture containing human cytochrome c and bacteriophage T7 RNA polymerase and identify each peak. (b) Sketch the results of SDS-PAGE of the same protein mixture showing the direction of migration and identifying each band.

(a) The ESI-MS spectrum below was obtained for hen egg-white lysozyme (HEWL). Using peaks 5 and 6, calculate the molecular mass of HEWL (see Sample Calculation 5-1).

[http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm. Published by A.E. Ashcroft, Astbury Centre for Structural Molecular Biology, University of Leeds (2000)]

(b) What is the charge on the ion that makes peak 5?

You are using ammonium sulphate to purify protein Q (pI = 5.0) by salting out from a solution at pH 7.0. How should you adjust the pH of the mixture to maximize the amount of protein Q that precipitates?

You wish to sequence the light chain of a protease inhibitor from the Brassica nigra plant. Cleavage of the light chain by trypsin and chymotrypsin yields the following fragments. What is the sequence of the light chain?

Chymotrypsin

1. Leu–His–Lys–Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser

2. Gln–Gln–Ala–Gln–His–Leu–Arg–Ala–Cys–Gln–Gln–Trp

3. Arg–Ile–Pro–Lys–Cys–Arg–Lys–Phe

Trypsin

4. Arg

5. Ala–Cys–Gln–Gln–Trp–Leu–His–Lys

6. Cys–Arg

7. Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser

8. Phe–Gln–Gln–Ala–Gln–His–Leu–Arg

9. Ile–Pro–Lys

10. Lys

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