You have produced the protein Glacate intracellularly in E. coli, lysed cells an

Question

You have produced the protein Glacate intracellularly in E. coli, lysed cells and clarified the solution which is now ready to be purified. The Glacate protein is relatively large (60 kDa) and positively charged (pI 9.7). On the surface of the Glacate there are three cysteines, which need to be free for the protein to be functional. The enzymatic activity of glacial enzymes is inhibited by zinc ions. You should now prepare the first purification step.

a) Name any material that the matrix could consist of.

b) Name any features that the matrix should have to fit.

c) What approximate size of the food rolls do you choose? Motivate!

d) Name any features that the mobile phase, the buffer, should have to passa.

e) Are there any additives that you may need to have in the buffer to facilitate cleaning of the Glacate?

Explanation / Answer

Chromatography is a labortory technique used to seperate differentcomponents of mixture. Mixture when dissolved in the liquid medium is called mobile phase, where as stationary pghase carries it through a structure which hold other material. In chromatography the various components of the mixture or cell lysate travels with different speeds, which causes mixture to seperate. However, the seperation is based on the partitioning between mobile and stationary phase. The chromatography technique follows perparative or analytical.

There are many types of chromatography with unique features such as, size exclusion (seperates mixtureon the basis of size), affinity (seperates mixtureon the basis of intractions between ligand-receptor, antigen-antiboy, enzyme-substrate), ion-exchange (seperates mixtureon the basis of pI). For the seperation of protein glacate present intracellulary in E. coli will seperate on the basis of ion-exchange or affinity chromatography which wholly depends on pI and intractions respectivly, of the cell lysate protein called glycate. As, pI of the glycate protein is 9.7 (pH-2.7). Therefore, ion-exchange chromatography would be used.

(1). The matrix for cation exchanger would be dextran or polystyrene which is hydrophobic porous polymer.

(2). The functional group for strong cation-exchanger will be sulphomethyl (-CH2CH2SO3-) or sulphopropyl (-CH2CH2CH2SO3-) which is strongly acidic in nature.

(3). The size of matri used in the ion exchange chromatography is 30-75 m.

(4). The mobile phase of cation exchange chromatography are aqueous containing diluted acids HCl, HNO3, H2SO4, or CH3SO3H which donate H+ in the column. The main feature for the buffer which were used in the ation exchange chromatography is its basic property.

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