Design of affinity tags for one-step protein purification from immobilizedzinc columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depend s on the chelated metal used and the elution profile of the host cell prote ins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to eit her immobilized copper. or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli p roteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very litt le cellular protein emerging at pH values lower than 7.0. Thus, a large por tion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins t o different regions of the elution profile, facilitating purification under mild conditions.