Question

You want to express a human protein in E. coli that has been transformed by a DNA plasmid containing the gene sequence for this protein. The sequence of the protein (in one-letter code) that will be expressed in E. coli is: MAGSGRLVLRPWIRELILGSETPSSPRAGQLLEVLQDAEAAVAGPSHAPDTSDVGATLLVSDGTHSV RCLVTREALDTSDWEEKEFGFRGTEGRLLLLQDCGVHVQVAEGGAPAEFYLQVDRFSLLPTEQPRL RVPGCNQDLDVQKKLYDCLEEHLHHHHHHHHHH

A. You proceed to express the protein, lyse open the cells, and remove the insoluble debris. With the soluble fraction of the lysate that contains your protein, you proceed to perform affinity chromatography. What specific kind of affinity chromatography (discussed in lecture) will you perform? Give a reason for your answer

B. You elute your protein off the affinity column and analyze a small amount of the eluted fraction on a coomassie-stained SDS-PAGE. You notice a band that corresponds to the size of your protein. But you find several other bands too in the elution fractions containing your protein. So, you decide to do ion-exchange chromatography on the impure protein using a buffer system at pH = 7. What specific kind of ion-exchange chromatography will you perform? Give a reason for your answer

Answer 1

A. With the soluble fraction of the lysate that contains your protein, we wish to perform Affinity Chromatography (His tag). A common technique involves artifically synthesizing a strect of 6 to 8 histidine residues into the N or C terminal of recombinant protein. This polyhistidine tail binds very strongly to nickel and cobalt metal ions and then it is passed through a column which has got a coating of immobilized nickel ions which binds the tag of polyhistidine. The protein is further eluted.

B. Impure protein will further be subjected to cation and anion exchange chromatography. Ion exchange chromatography is considered to be very powerful technique in the field of protein purification. Cation exchange column have positively charged counter ions and compounds which are positively charged present in a mixture which is further passed through a column. Exchange of counter ions takes place and binds to negatively charged groups.

Anion exchange column , the groups are attached to positively charged beads and negatively charged are counter ions.