Genetic system and self-cleaving inteins derived therefrom

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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. The enhanced splicing mutant has utility in purification of proteins such as toxic proteins, for example, by inactivation with the intein in a specific region and controllable splicing. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing. Thus, disclosed and claimed are: a genetic system and self-cleaving inteins therefrom; bioseparations employing same; protein purification by inactivation with inteins in specific regions and controllable intein splicing; methods for determining critical, generalizable residues for varying intein activity; and products.

In process biotechnology, purification of proteins from complex biological mixtures involves a series of complicated recovery steps, each of which can compromise the purity and yield of the desired product Fish et al. (1984) BioTech. 2:263.

Reducing the number of such unit processes and their complexity would significantly improve product purity and yield while reducing costs. Fusion based affinity separations provide a simple means of isolating target proteins from complex cell extracts by making use of highly specific interactions between fused peptides and small, easily immobilized ligands. LaVallie et al. (1995) Curr. Opin. Biotechnol. 6:501-506; and Linder et al. (1998) Biotech. Bioeng. 60:642-647. Although fusion-based affinity systems have been known for some time and used extensively in the laboratory, their limitations have precluded their wide use in large scale applications.

In the conventional technique, the DNA coding sequence of a target protein is joined to the DNA sequence of one of a number of binding proteins to form a single open reading frame. Expression results in a two-domain fusion protein that can be easily purified via the affinity of the binding domain for its immobilized ligand. The use of optimized affinity resins minimizes the nonspecific binding of contaminant proteins, ensuring that the fusion product is recovered at high purity. Following purification, the target protein is cleaved from the binding domain at the fusion joint, where the recognition of an appropriate protease has been inserted. The product stream of this purification is a relatively simple mixture consisting of the highly purified protein of interest, the cleaved binding domain, and a small amount of protease.

The potential of this technique for use in large scale pharmaceutical production is limited in part by complications arising from the addition of protease to the purified fusion protein solution. The primary limitation is nonspecific cleavage within the product protein by the protease, leading to the destruction of the desired protein. A second disadvantage is cost; as scales increase, more protease is required, dramatically increasing production costs. Finally, the addition of protease necessitates an additional purification step, and can complicate drug approval due to the highly bioactive nature of these enzymes.

A recent advance in this area has been the introduction of self-cleaving protein linkers, achieved by combining binding domains with modified self-splicing protein elements known as inteins. Discovered in 1990, inteins are naturally occurring internal interruptions in a variety of host proteins. Hirata et al. (1990) J. Biol. Chem. 265:6726-6733; Kane et al. (1990) Science 250:651-657; Perler et al. (1994) Nucl. Acids Res. 22:1125-1127; and Noren et al. (2000) Angew. Chem. Int. Ed. 39:450-466.

Following translation of the host protein-intein precursor sequence, the intein excises itself and ligates the flanking host protein segments (exteins) to form the native host protein and released intein. A major advantage of the claimed method is that the cleavage reaction can take place on the column, eliminating the need for any further purification. Additionally the cleavage reaction only affects the target protein, thus, nonspecifically bound contaminant proteins are not affected and are not released into the product stream. This strategy forms the foundation of the commercially available IMPACT-CN system (New England Biolabs, Beverly, Mass.). (FIG. 1A). Perler et al. (1994). Because the structural information required for splicing exists entirely within the inteins they can be used in a variety of applications involving intein insertion into foreign contexts. The ability to construct intein fusions to proteins of interest has broad potential application. Gimble (1998) Chemistry & Biology 5:R251-R256. One of these is affinity fusion-based protein purification, where an intein is used in conjunction with an affinity group to purify a desired protein. Chong et al. (1997b) Gene 192:271-281; and Chong et al. (1998b) Nucl. Acids Res. 26:5109-5115. Self-cleavage, rather than splicing of the intein releases the desired protein (FIG. 1B), thereby eliminating the need for protease addition and simplifying overall processing. However, this system has drawbacks. First, in the configuration where the product protein is released by N-terminal cleavage, the cleavage reaction requires the addition of thiol containing compounds that modify the C-terminus of the product protein. Native protein is recovered only after subsequent hydrolysis of the cleavage-inducing reagent. Chong et al. (1997a) J. Biol. Chem. 272:15587-15590. Second, where the product protein is released by C-terminal cleavage in the IMPACT-CN system, the reaction is accompanied by unwanted N-terminal cleavage, requiring the N-terminal fragment to be removed in an additional purification step (described in product literature). Third, the large size of the 56-kDa Saccharomyces cerevisiae intein in the IMPACT system can diminish solubility and purification efficiency. For this application to be more attractive, the intein must be altered to yield optimized controllable cleavage rather than splicing. Furthermore, the intein should be as small as possible for this strategy to be attractive for scaleup.

Recent studies have determined that large inteins are bipartite elements consisting of a protein splicing domain interrupted by an endonuclease domain. Dalgaard et al. (1997a) Nucl. Acids Res. 25:4626-4638; Duan et al. (1997) Cell 89:555-564; and Derbyshire et al. (1997a) Proc. Natl. Acad. Sci. USA 94:11466-11471. Because endonuclease activity is not required for protein splicing, mini-inteins with accurate but reduced splicing activity can be generated by deletion of this central domain. Derbyshire et al. (1997b); Chong et al (1997a); and Shingledecker et al. (1998) Gene 207:187-195. Mechanistic studies have also determined the roles of highly conserved residues near the intein/extein junctions in the splicing reaction (FIG. 1A). Chong et al. (1996) J. Biol. Chem. 271:22159-22168; Xu et al. (1996) EMBO J. 15:5146-5153; and Stoddard et al. (1998) Nat. Struct. Biol. 5:3-5. These residues include the initial Cys, Ser or Thr of the intein, which initiates splicing with an acyl shift, the conserved Cys, Ser or Thr immediately following the intein, which ligates the exteins through nucleophilic attack, and the conserved C-terminal His and Asn of the intein, which release the intein from the ligated exteins through succinimide formation. Mutation of these residues can be used to alter intein activity to yield isolated cleavage at one or both of the intein-extein junctions. Chong et al. (1998b) J. Biol. Chem. 273:10567-10577.

Despite insights into intein structure and function, modifications often resulted in unacceptably low activity, poor precursor stability, or insolubility. Derbyshire et al. (1997b); Chong et al. (1997b); Shingledecker et al. (1998); and Chong et al. (1998a).