Fast and cheap protein purification: Economical alternatives to conventional chromatography-based recombinant protein purification.

摘要

Two naturally produced in vivo affinity resin substitutes have been combined with a self-cleaving tag to develop two novel affinity-based purification technologies. The first resin alternative is polyhydroxybutyrate (PHB) granules and the second is elastin-like polypeptides (ELP). An engineered self-cleaving intein is combined in a tripartite fusion to form phasin-intein-target protein, for the PHB, or ELP-intein-target protein, for the ELP method. In the first case tagged product proteins are expressed in E. coli strains that produce intracellular PHB granules, where the fusions bind to the granules via the PHB-binding phasin tag. The granules and attached proteins can then be easily recovered following cell lysis by simple centrifugation. Once PHB-bound fusions are purified, a mild pH shift triggers intein self-cleavage and release of the product protein from the granules into solution. In case of the ELP technology, both resin and affinity tag are substituted by an ELP composed of many repeats of several amino acids (VPGXG). This protein structure has a unique and reversible temperature-sensitive solubility property. Expressed ELP-intein-target protein fusion separates from the soluble and insoluble cell components by a temperature increase or salt addition and centrifugation. Cooling or salt removal resolubilizes this fusion and intein cleavage is used in a similar fashion to release the product protein.; The two systems have been successfully used at laboratory scale to purify more than a dozen proteins varying in size, complexity and activity, demonstrating the proof of principle, high purity and yield attainable. Hence, the cost associated with purification of recombinant proteins is reduced significantly to effectively that of just the culture medium. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries. A plethora of options are possible as potential future extensions of this work.; Furthermore, a conventional chromatography column has been modified with a pH-controlled purification tool to yield a constant concentration peak in the purified column effluent. A mathematical model closely follows experimental data and is a basic tool for prediction and development of more complicated outputs.