A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

We introduce a new method for the purification of recombinant proteins expressed in Escherichia coli using self-cleaving elastin-like polypeptide (ELP) fusion tags without the need for affinity chromatography or proteolytic tag removal. Using this method we obtained high purity, activity and reasonable yields for ten diverse target proteins.

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.Inteins are protein-splicing elements that exist as in-frame fusions with flanking protein sequences called exteins. Inteins are self-splicing at the protein level, with their excision being coupled to extein ligation (1-3). Most of the inteins that have been described are in the 400-to 500-aa range with little absolute sequence conservation among the elements (4, 5). However, Cys or Ser residues are required at the amino termini of both the intein and the second extein, and a His and Asn are present at the carboxy terminus of the intein (Fig. 1A Top). Most inteins contain eight conserved sequence blocks (A-H), two of these being the LAGLIDADG motifs (blocks C and E) that define a family of intron-homing endonucleases (refs. 4 and 5; Fig. 1 A). Consistent with the occurrence of these motifs, several inteins have been shown to have site-specific endonuclease activity (6), and PI-SceI, the VMA1 intein of Saccharomyces cerevisiae, is capable of homing into a cognate inteinless allele (7). The sporadic distribution of inteins in all three biological kingdoms is consistent with their being mobile elements.Endonuclease genes have been assumed to be invasive genetic elements that colonized group I introns, converting them into mobile genetic elements (8-11). Similarly, mobile inteins appear to be derived from invasive endonuclease genes. Recent structural studies indeed suggest that the proteinsplicing and endonuclease domains are separate and that their two activities may have evolved independently. First, the crystal structure of PI-SceI has recently been solved (12). This 454-aa protein is folded into two distinct structural domains. Second, hidden Markov models have been used to define two conserved functional domains of inteins, corresponding to independent endonuclease and splicing modules, separated by nonconserved spacer regions of variable lengths (ref. 13; J.Z.D., A. Klar, M. J. Moser, W. R. H...