You have identified a protein in humans with the amino acid sequence shown here:

Question

You have identified a protein in humans with the amino acid sequence shown here:

1 - M Q I F V K T L T G K T I T L E V E P S D T I E N - 25

26 - V K A K I Q D K E G I P P D Q Q R L I F A G K Q L - 50

51 - E D G R T L S D Y N I Q K E S T L H L V L R L R G – 75

76 - G

A. Estimate the molecular weight for this protein. Explain your reasoning and show your work.

B. Estimate the pI for this protein. Explain your reasoning and show your work.

C. How many disulfide bonds do you propose this protein contains? Explain your reasoning.

D. What form of ion-exchange chromatography would you use to purify this protein at a pH of 9.3? Explain your reasoning.

E. Another researcher proposes that the function of this protein is regulated by phosphorylation. Do you think this hypothesis is plausible? Explain your reasoning.

Explanation / Answer

A. Theoretical Mw: 8564.84 dalton (sum of molecular waight of AA)

B. Theoretical pI: 6.56 (Net Sum of charges on AA) (you can do isoelectric focussing using ampholines (gel) of different pH ranges in order to get the correct value of the isoelectric point (pI).

C. 0, A valid amino-acid sequence must contain at least two cysteines to form the Disulfide bond.

D.If you want to use it for purification purpose using ion exchange chromatography. If pH of the buffer is higher than pI , protein will bear negative charge due to deprotonation of these groups. In order to get complete deprotonation of these ionizable groups, pH should be 2 units higher than pI of the protein. Therefore, you can generate net negative charge on the protein and select weak anion exchanger (DEAE-Sephadex) if protein is negatively charged for efficient binding of your protein to the ion exchanger.

E.Possible, the amino acids most commonly phosphorylated are serine, threonine, and tyrosine in eukaryotes, which play important and well-characterized roles in signaling pathways and metabolism. This protein contains this residues.

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