Introduction Results

Introduction

Split-inteins can be used to simplify conventional protein purification in many aspects: a split-intein based system is cheaper, faster and more efficient than other purification procedures. In fact, only a single chromatographic step is necessary for both, binding of the protein fused to an affinity tag to the affinity column and tag removal. Also, there is no need for expensive proteases. We standardized this split-intein purification system, so it is easy applicable to all kinds of proteins.

In previous purification procedures, there a several steps to take to get solely the purified protein: The first step is the binding of the protein to the affinity matrix, the washing and finally release of the protein from the affinity column, the protein still being bound to the affinity tag. The next step is the removal of the affinity tag from the protein by protease cleavage, which causes undesirable secondary cleavage within the protein sequence. In industrial scales proteases are the most expensive component during the purification process. Finally, the split off affinity tag has to be separated from the protein, which takes again a lot of effort.

The split-intein based system is manifold easier: All these steps can be combined into one by using Ssp DnaB mini-intein, an artificial split intein. It performs the trans-splicing reaction only under certain pH and temperature conditions. A mutation of the SspDnaB N-intein at Cys1 to Ala prevents cleavage at the N-terminus, cleavage at the C-terminus still occurs. The N-terminal intein part, attached to a chitin binding domain can be used as an affinity ligand, while the C-terminal part, fused to a protein of interest, can be used as an affinity tag binding highly specific to the ligand part. After loading the protein of interest on a chitin column, the trans-splicing reaction can be induced by adjusting temperature and pH: the both split inteins fuse together, disrupting the fusion to the protein of interest in one single step [1].

References

[1] Lu, Wei et al.: Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage. J. Chromatogr. A 1218 (2011) 2553–2560. DOI: 10.1016/j.chroma.2011.02.053.