You have a mixture of the following proteins: Protein A, 25 kDa, pl 5.4 Protein

Question

You have a mixture of the following proteins: Protein A, 25 kDa, pl 5.4 Protein B. fused with a His tag. 140 kDa, pl 5.2 Protein C, 23 kDa, pl 9.5 Protein D, 60 kDa, pl 8.2 Protein E, fused with an MBP tag. 100 kDa, pl 4.9 Describe in detail how you would separate protein B from this mixture using column chromatography approaches. Include in your description any required columns, reagents, and buffers, and the physical basis for the separation technique. You are working with a protein, and find that it has a glutamate residue surrounded by nonpolar residues in the protein interior. a) Please provide the 3-etter codes for 3 amino acids with predominantly nonpolar side chains that might be found in this pocket. b) would you expect the pka of the glutamate side chain described in this question to be higher, lower, or the when compared to a glutamate side chain found on the protein Please explain your reasoning.

Explanation / Answer

Separation of Protein B from mixture

The protein from the mixture can be separated using gel electrophoresis, which separates proteins based on their molecular weights. Protein B has highest molecular weight ad is thus convenently separated from the rest of the proteins using column chromatography.

The higher molecular weight polymer comes out first from the column leaving the rest behind.

1. For the given pockets

a. Non-polar amino acids would be,

Gly, Ala, Val, Leu, Met, Ile, Phe, Trp, Pro

b. The glutamate would have the same pKa value as the non-polar interactions are least strong and thus not affect the pKa values.

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