Question

Molecular weight: 93910.16 Theoretical pI: 6.03 1.

1. Based on the pI of Taq, what pH buffer would you have to use to get Taq polymerase to stick to an anion exchanger?

Briefly discuss the advantages and disadvantages of using a salt or pH in eluting proteins from an ion-exchange column.

Please place details explaining how you arrived to ech answer for question 1

Answer 1

Given

Taq protein: - Molecular weight: 93910.16 Theoretical pI: 6.03

When the pH is lower than the pI, the amino acid will carry a net positive charge. When the pH is higher than pI, then the amino acid carries a net negative charge.

We need to use anion exchanger therefore molecule must be negative. Then we must use higher pH buffer. Generally we use 0.5-1.5 pH units more than pI. Therefore, pH need to be 6.53-7.53 to get Taq polymerase to stick to an anion exchanger.

Protein is eluted using either using the salt or changing pH.

Charged salt ions compete for resin functional groups with bound proteins and helps eluting the protein. pH gradient is chosen according to the pI of protein so that protein will have net neutral charge. Then protein will elute from column.

Salt elution method is widely used in ion exchange chromatography. Salt does not modify the structure of protein in irreversible manner like pH gradient method. We can increase the ionic strength of salt to elute proteins according to binding characteristics (more or less charged groups- weak or strong binding). Salt elution is generally done in steps.

pH gradient method is less common. Chemical stability must be thoroughly studied before performing pH gradient elution as it can denature protein. pH gradient is fast process as compared to salt elution. If we want to elute Taq protein(pI=6.03) we can easily prepare buffer with same pH and elute desired protein.