Question

You are given a sample containing two proteins in phosphate-buffered saline: Protein A (pI value 8.4) and Protein B (pI value 7.0).

You wish to separate this protein mixture by ion-exchange and have available a column of CM­Sephadex. To carry out the procedure, you have prepared an appropriate MES buffer (50 mM, pH 6.0, with 20 mM NaC1). We will call this Buffer M.

(i) Suggest a preparatory procedure and materials needed to change the phosphate buffer of the protein sample with the MES buffer (Buffer M) required for ion-exchange.

(ii)Once the proteins are introduced into Buffer M, comment on the overall charge states for each of Protein A and Protein B.

(iii)The column of CM-Sephadex is equilibrated with Buffer M. What is the overall charge state of the matrix under these buffer conditions?

(iv)The protein mixture is now introduced to the column. Describe the process of adsorption that will occur. Which of the two proteins, Protein A or Protein B, will experience strongest adsorption under these conditions? Justify your answer.

(v) What should be done next to effect the best separation of Protein A and Protein B from the matrix? Give a full molecular description of events.

(vi) Consider a different separation procedure in which a different buffer choice was made to carry out the separation on CM-Sephadex. In this second scenario, Buffer T, containing 50 mM Tris buffer, pH 7.5 with 50 mM NaC1 was used for starting conditions. Outline how a separation of Protein A and Protein B can still be effected in Buffer T utilizing CM-Sephadex matrix. Explain the order of elution that would occur for each of the Protein A and Protein B species with your new procedure.

(vii) Comment on whether it would be possible to separate Protein A from Protein B again using Buffer T, but with a different column chemistry, one containing Mono-Q matrix.

Answer 1

1. For ion exchange your protein should be in the same buffer so you have to dialyze your protein in same buffer. For that buffer and dialysis membrane is required of some instrument that can exchange the buffer.
2. In this case pH< pI for both the case. In buffer M both protein contain positive charge.
3. As CM-sepharose is weak ion-exchanger and it take negative charge at this pH.
4. In this chromatography protein is positively charged and CM-sepharose is negatively charge, therefore they make ionic interaction. In this pI and pH difference between protein is high that indicate more positively charged protein is present such as( that’s why pI is high) such as lysine and arginine. Hence protein A binds more strongly.
5. Increase in pH and increase in salt (NaCl) will be carried out for separation. These two strategy can be apply first we increase the pH up to 7 so pI 7 protein will come out due to zero charge on it then again the increase the pH then pI 8.4 protein will come out. In second approach increasing sodium chloride gradient will elute the protein. As the concentration of NaCl will increase then it breaks the ionin interaction between protein and CM sepharose and protein will elute.
6. If starting buffer having pI 7.5 than protein B will be negatively charged and it will not bind whereas protein A will bind. Protein B come in flow though and second protein will elute by increasing pH or by increasing salt concentration.
7. As in buffer T, protein B will become negatively charge (pH>pI) but protein A is still positively charge. Here mono Q (Quaternary amino ; strong ion-exchanger) will bind with negatively charged protein. Here protein B, negatively charged, will bind but positively charge protein A will not bind.