Purification of E. coli proteins using a self-cleaving chitin-binding affinity tag

Abstract

The use of affinity tags to purify recombinant proteins is ubiquitous in molecular biology. However, tag removal after purification still remains a challenge. The most commonly used method, proteolytic digestion, has several drawbacks that make the process complex and costly. One alternative to the use of proteolytic digestion is the use of self-cleaving purification tags. Here, we describe a system that combines a chitin-binding domain (CBD) tag with the ∆I-CM intein to yield a self-cleaving purification tag. A protein gene of interest is genetically fused downstream of the tag, generating a fusion protein that can be rapidly and easily purified using a chitin resin. Intein self-cleavage is then induced by a simple pH and temperature shift, liberating the free target protein. This system can be used to readily purify any recombinant protein that can be expressed in E. coli, and has the potential to be applied to a wide variety of additional tags and expression hosts.