Proteins from PHB granules

Abstract

Over the years a number of techniques that rely on the fusion of a gene to a sequence encoding a polypeptide that can be readily purified by affinity chromatography have been devised to facilitate the protein purification both at the analytical and at the industrial scale (Nilsson et al. 1997; Hearn and Acosta 2001; Jonasson et al. 2002). While these techniques had greatly expedited the production of cloned products, they are not without drawbacks. First, affinity chromatography matrices are quite costly, an issue that becomes particularly significant in large-scale applications (Hearn and Acosta 2001; Przybycien et al. 2004). Second, the fusion protein has to be proteolytically cleaved and the affinity tag must be separated from the desired polypeptide. A number of strategies to circumvent the cost of affinity chromatography or to facilitate the proteolytic cleavage of the fusion protein have been developed. For example, Meyer and Chilkoti (1999) reported the first nonchromatographic method, termed inverse transition cycling, for recombinant protein purification by fusion with a thermally responsive polypeptide (elastin-like polypeptide [ELP]). ELP consists of repeats of the pentapeptide sequence and undergoes a reversible inverse temperature transition. When the temperature is raised above the inverse transition temperature (Tt), the normally soluble ELP fused polypeptides form insoluble aggregates which can be separated by simple centrifugation. Subsequently, the aggregate can be resolubilized easily by a temperature shift below Tt. Recently, Chen's group (Stiborova et al. 2003) introduced affinity-modified ELPs, including imidazolegrafted ELPs for the precipitation of His-tagged proteins, thus providing an effective alternative to immobilized metal affinity chromatography. Along similar lines, Mattiasson's group (Kumar et al. 2003) reported new affinity precipitation reagents based on the thermoresponsive polymer poly(N-isopropylacrylamide) modified with vinylimidazole metal ion chelating groups to generate thermoresponsive metal affinity precipitants. At the other end, the intein technology enables the selfcleavage of protein fusions. Inteins are the protein analogs of self-splicing RNA introns, as they posttranslationally excise themselves from various precursor proteins. The ability of inteins to catalyze a precise peptide bond cleavage in a controllable manner can eliminate the use of protease for the removal of a fusion tag during the recovery steps and also a subsequent protease removal step which potentially reduces the cost of a large-scale purification process for a recombinant protein product. In this issue of Protein Science, Banki et al. (2005) have now pushed the frontiers of gene fusion technology several steps forward. In this technology, precipitation of intein fusion proteins is accomplished by affinity binding to insoluble particles produced by the cell itself. Following precipitation, self-cleavage of the intein results in the release of the purified polypeptide. Banki et al. (2005) capitalized on phasins, proteins that bind tightly to polyhydroxybutyrate (PHB) granules that can be readily separated from lysed bacteria. In their strategy, cells are first engineered to produce PHB granules to which the phasin–intein fused target protein binds. Following cell lysis, the PHB granules are separated by centrifugation, and the soluble target protein is recovered following the intein-mediated self-cleaving reaction that is triggered by adjusting the pH. Banki et al. (2005) reported the purification of several recombinant proteins including maltose binding protein, β-galactosidase, chloramphenical acetyltransferase, and NusA, with yields ranging from 30 to 40 mg/L culture. Polyhydroxyalkanoates, including PHB, are storage polymers produced by various bacteria when the culture environment is not optimal for growth. During the past decade, polyhydroxyalkanoates have attracted commercial and academic interest as a