Protein Purification in mixture contains proteins A, B and C. The MW values belo

Question

Protein Purification in mixture contains proteins A, B and C. The MW values below are for a single protein chain. 8. A prote Protein A-pl 5.5, Mw = 50200 Da, homodimer Protein B-pl-7.5, MW- 35000 Da, monomer Protein C-pl-8.5, MW 65300 Da, monomer (a) In which order will proteins A, B and C elute from a gel filtration column? Explain your answer. (b) Which protein will salt out the easiest at pH 7.5? Explain your answer. (c) Describe how you could perform ion exchange to separate protein A from proteins B and C?

Explanation / Answer

In gel filtration chromatography protein molecules are separated on the basis of their size. The colum consist of porous beads ,large MW proteins are excluded from moving into the pores and small molecular weight protein/size than the pores can enter into them. Thus big molecules are eluted first and small molecules have a longer transit time and are eluted last.

Protein A is a homodimer and the molecular weight of its single chain protein (monomer) is 50200 Da. The total size of protein A is 50200 +50200 = 100400 Da. Protein B and C are monomers thus their size 35000 and 65300 dalton respectively.

Decreasing order of size is Protein A > Protein C > Protein B

Protein A will elute first then protein C and protein B will elute out in the last.

b) At ph 7.5 protein B will be neutral as its pI (isoelectric) value is 7.5 and when pI = pH net charge on proteins is zero. Proteins are soluble in water due its interaction with water molecules via its charged surface. At isoelectric point proteins are least soluble in water. The absence of net positive or negative charge on the surface would prevent electrostatic repulsion between protein molecule that keeps them solubilized in water. Absence of charge on protein will cause them to interact with other protein molecule and form aggregates leading to precipitation out of water.

c) Protein A with a pI of 5.5 will be negatively charged at a ph of 7.5. Protein B will be neutral and Protein C will be positively charged. First mixture of proteins is mixed with equlibriation buffer. When this mixture is applied to a colum containing anion exchanger DEAE- cellulose, which has positively charged amino group, equilibriated with ph7.5 buffer, protein A will bind because it is negatively charged at ph7.5 . Protein B and C will elute becuase B would not be able to bind as it lacks any charge and C would be repulsed from the exchanger because it is positively charged at ph 7.5.

Column bound protein A can be eluted by increasing salt concentration or altering pH.

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