Protein A 65(kDa) pI (7.2) - glycoprotein Protein B 72(kDa) pI (7.8) - antibody

Question

Protein A   65(kDa) pI (7.2) - glycoprotein

Protein B   72(kDa) pI (7.8) - antibody available; not glycosylated

Protein C   63(kDa) pI (6.8) - not glycosylated

Protein D 70(kDa) pI (8.8) - glycoprotein

Giving the above information on protein components of a medical sample from a human patient that is to be purified :

(a) Propose a suitable series of chromatography steps to separately isolate each of the four protein components of this mixture, whilst maintaining their biological fold. At least one of the series of steps should involve affinity chromatography, present your purification protocol as a flow chart, outlining the types of chromatography you suggest, and the chromatography media required.

(b) Select an affinity step in your procedure. Prepare a full set of instructions for carrying out the step in the protein laboratory. Give chemical names for the chromatography media, buffer system and any salts needed. Include concentrations as well.

Explanation / Answer

1) Separation steps involves-

                                             Size exclusion chromatography ( to separate protein on the basis of their size) by using sephadex G-75 (which separates protein containing weight 3kd to 70 kda).

                                        Affinity chromatography which will separate protein B since for this protein antibodies are available. Lectin affinity chromatography can also be used here to separate them according glycol protein.                             

                                       Ion exchange chromatography which will separate them according to difference in their pH.

2)

1.The method used to separate glycoprotein and non-glycoprotein forms is lectin affinity chromatography. But in some cases Lectin affinity resins have a low binding capacity.

2. Reverse phase chromatography can also be used but this also have drawbacks in the separation of glycoprotein and non-glycoproteins since it relies upon the use of high concentrations of acetonitrile and may also utilize strong acids such as trifluoroacetic acid (TFA). This may result in adverse effect upon protein function.

3. Glycoprotein and non-glycoprotein forms can be separated on an S Sepharose High Performance column in the presence of 5% ethylene glycol/5% n-butanol.

4. Another method for separating glycoprotein from non-glycoprotein is-:

This involves different steps-

(a) equilibrating a solution containing Glycoprotein and non-glycoprotein forms with an equilibrating solution containing at least alcohol and glycol (concentration 50%);

(b) adding the equilibrated protein solution onto a chromatographic column containing a cationic or anionic packing for the ion exchange separation of Glycoprotein and non-glycoprotein forms;

(c) eluting the glycoprotein from the chromatographic column substantially free of non-glycoprotein using a first eluting solution as written above;

(d) eluting the non-glycoprotein from the chromatographic column substantially free of glycoprotein using a second eluting solution (at least one C1-C4 alcohol and/or C1-C4 glycol in a concentration of below 50% per total volume and optionally (ii) at least one salt);

            (c) and (d) optionally being reversed.

At last both protein are separted.

                               Or

Con A-Sepharose and WGA-agarose are used while using lectin affinity chromatography.

Related Questions

Navigate

• Browse (All)
• Browse P
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.