Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is b...

Protein splicing is a post-translational process in which a biologically inactive protein is activated by the release of a segment denoted as an intein. The process involves four steps. In the third, the scission of the intein takes place after the cyclization of the last amino acid of the segment, an asparagine. Little is known about the chemical reaction necessary for this cyclization. Experi...

Protein splicing is an intricate self-catalyzed protein rearrangement that converts an inactive protein precursor to biologically active proteins. In the past decade, mechanistic studies and extensive engineering of the naturally occurring protein splicing elements, termed inteins, has led to the development of numerous novel technologies. These intein-based methodologies permit in vitro and in...

‘‘Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a regulatory role for splicing . . . ’’ (1). That statement was made more than a decade ago by our colleagues and ourselves in reference to the phage T4 group I introns, all three of which reside in genes involved in nucleotide metabolism. The intervening years were distinguished by...

BACKGROUND Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E....

Acyl shifts involving N-S and S-S rearrangements are reactions central to the breaking of a peptide bond and forming of thioester intermediates in an intein-catalyzed protein splicing that ultimately leads to the formation of a new peptide bond by an uncatalyzed S-N acyl shift reaction. To mimic these three acyl shift reactions in forming thioesters and the subsequent peptide ligation, here we ...

Abstract Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for one‐pot semi‐synthesis modified proteins. EPSL harnesses rapid kinetics reactions between synthetic selenopeptides aryl selenoesters (generated from intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentra...