Abstract
BackgroundBinding of proteins in ion exchange chromatography is dominated by electrostatic interactions and can be tuned by adjusting pH and ionic strength of the solvent. Therefore, the isoelectric region (IER), the pH region of almost zero charge near the pI, has been used to predict the binding properties of proteins.Principal findingsUsually the IER is small and binding and elution is carried out at pH values near to the pI. However, some proteins with an extended IER have been shown to bind and elute far away from its pI. To analyze factors that mediate the size of the IER and to identify proteins with an extended IER, two protein families consisting of more than 7000 proteins were systematically investigated. Most proteins were found to have a small IER and thus are expected to bind or elute near to their pI, while only a small fraction of less than 2% had a large IER.ConclusionsOnly four factors, the number of histidines, the pI, the number of titratable amino acids and the ratio of acidic to basic residues, are sufficient to reliably classify proteins by their IER based on their sequence only, and thus to predict their binding and elution behaviour in ion exchange chromatography.
Highlights
Ion exchange chromatography (IEC) is a widely applied method in protein purification
Only four factors, the number of histidines, the pI, the number of titratable amino acids and the ratio of acidic to basic residues, are sufficient to reliably classify proteins by their isoelectric region (IER) based on their sequence only, and to predict their binding and elution behaviour in ion exchange chromatography
A large IER has been shown to influence the binding of proteins to ion exchange columns which has been demonstrated for the lipase B from Candida antarctica [9]
Summary
Ion exchange chromatography (IEC) is a widely applied method in protein purification. A detailed investigation of pH values at which bound proteins eluted from an anion exchange chromatography column were performed using a pH gradient as the method of elution. It demonstrated that for proteins with pI values between 6 and 8, the elution occurred at pH values considerably higher than their pI. A large IER has been shown to influence the binding of proteins to ion exchange columns which has been demonstrated for the lipase B from Candida antarctica [9]. The purification of this protein by ion exchange chromatography had not been achieved before. Most proteins were found to have a small IER and are expected to bind or elute near to their pI, while only a small fraction of less than 2% had a large IER
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