Protein transSplicing and Cleavage Systems

The N- and C-terminal segments of an artificially or naturally split intein, each segment being fused to a foreign protein sequence, are able to assemble and mediate protein trans-splicing, yielding a native peptide bond between the two foreign protein sequences (Lew et al. 1998; Mills et al. 1998; South-

worth et al. 1998; Wu et al. 1998a,b; Yamazaki et al. 1998; Evans et al. 2000). In particular, studies of a naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein; Wu et al. 1998a) have led to its use in protein ligation, cyclization and purification. It has been shown that the N-terminal 123-residue segment and the C-terminal 36-residue segment of the Ssp DnaE intein associate with high affinity and mediate proficient ligation of two target protein sequences through a native peptide bond in vivo or under mild conditions in vitro (Evans et al. 2000; Martin et al. 2001). The trans-splicing approach has been employed to split a target gene in two halves for expressing the target gene into two primary translational products. The functional target gene product can be subsequently reconstituted in vivo and in vitro. One successful example is the reconstitution of herbicide resistance from a split gene encoding 5-enolpyruvylshkimate-3-phosphate synthase (EPSPS) in transgenic plants (Chin et al. 2003). Another utilization of the trans-splicing approach is in vivo cyclization of proteins and peptides, which aims to produce presumably more stable circular polypeptides in cells. The split intein-mediated circular ligation of peptide and proteins (SICLOPPS) was achieved by sandwiching a target protein or peptide between the C- and N-terminal intein halves such that protein splicing during protein expression ligates the two termini of the target protein/peptide, yielding circular proteins or peptides in vivo (Scott et al. 1999; Evans et al. 2000).

The frans-splicing activity can be modulated to a frans-cleavage activity by employing a similar mutational strategy as that of the czs-splicing inteins (Chong et al 1997; Nichols et al. 2003). For instance, changing the last Asn residue of the C-terminal fragment of the split Ssp DnaE mini-intein to Ala converts a frans-splicing system into a frans-cleavage system (Fig. 5). A target protein fused to the N-terminal fragment can be expressed as a stable product. Addition of the intein C-terminal fragment containing the Asn-to-Ala mutation led to the reconstitution of the intein cleavage activity and, under inducing conditions, the release of the target protein. Use of the trans-cleavage fusion system may avoid the problem of in vivo premature cleavage of the intein-tag occasionally encountered by other intein fusion systems.

Fig. 5. Protein frans-cleavage utilizing the split Ssp DnaE intein. a diagram illustrating protein frans-cleavage. The protein of interest is fused to the intein N-terminal 123 amino acid residues, In(N). The intein C-terminal fragment of 36 amino acid residues, In(C), carries a substitution of the intein's last residue, Asnl59 with an Ala, which blocks splicing activity and cleavage at the C-terminal splice site. The fusion of a small chitin-bind-ing domain (CBD) to each intein segment facilitates the isolation of the expressed intein

Protein Cleavage

fusion proteins by binding to chitin resin. Protein frans-cleavage is initiated by mixing the two chitin-bound protein fractions, resulting in the reconstitution of a cleavage-proficient intein under native conditions. The target protein is released upon overnight incubation at

4 °C in column buffer containing 20 mM Tris-HCl (pH 7.0), 0.5 M NaCl and 30 mM DTT. The frans-cleavage product can be used for IPL by the replacement of DTT with 2-mer-captoethanesulfonic acid (MESNA). b Example of protein frans-cleavage resulting in single chitin column purification of maltose-binding protein (MBP).MBP-In(N)-CBD fusion protein and CBD-In(C)-CBD fusion protein were expressed in E. coli from pMEB14 and pBEB, respectively, and each was isolated by chitin resin. The cleavage reaction was initiated by mixing the chitin-bound protein fractions in the presence of 30 mM DTT at 4 °C. Samples taken from different purification steps were analyzed by 12% SDS-PAGE stained with Coomassie Blue. Lane 1 Induced cell extract of MBP-In(N)-CBD (MEB); lane 2 clarified cell extract of MEB; lane 3 MEB extract after passage through a chitin column; lane 4 chitin-bound MEB after washing with a column buffer (10 mM Tris-HCl, pH 8.5,0.5M NaCl); lane

5 induced cell extract of CBD-In(C)-CBD (BEB); lane 6 clarified cell extract of BEB; lane 7 BEB extract after passage through a chitin column; lane 8 chitin-bound BEB after washing with column buffer; lane 9 a fraction of mixed MEB and BEB; lane 10 a fraction of the mixed chitin resin after the 4 °C overnight incubation in the presence of DTT; lane 11 the supernatant containing the MBP product after centrifugation of the chitin resin shown in lane 10

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