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Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional...

In the past 15 years, nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology (1, 2). It not only is capable of solving protein structures to atomic resolution but also has the unique ability to accurately measure the dynamic properties of proteins and to probe the process of protein folding (3, 4). However, a major drawback of macromol...

The development of self-cleaving fusion-tag technology has greatly simplified the purification of recombinant proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods at a variety of scales. In this review, we describe some of these methods, and provide a ru...

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their ...

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two separated halves, and the recognition and association of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodynamic analysis on ...

We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this proce...