Siclopps

By rearranging the order of the elements in the Ssp DnaE frans-intein, an active czs-intein (C-terminal intein:target peptide:N-terminal intein) was obtained that upon splicing resulted in cyclization of the target protein (Scott et al. 1999; Evans et al. 2000). Named split intein-mediated circular ligation of peptide and proteins (SICLOPPS; Scott et al. 1999; Abel-Santos et al. 2003), this method allows for the genetic encoding and in vivo production of cyclic peptides and proteins. After translation of the C-intein:peptide:N-intein fusion, in vivo formation of a peptide bond between the first and last amino acids of the target sequence is catalyzed by the reconstituted intein, thereby creating a cyclic peptide or protein (Fig. 3).

SICLOPPS was used to cyclize the E. coli enzyme dihydrofolate reductase (DHFR; Scott et al. 1999). DHFR had been previously circularly permuted (Buchwalder et al. 1992; Uversky et al. 1996; Iwakura and Nakamura 1998) and cyclized via a disulfide bond (Iwakura and Honda 1996). A three amino acid linker (or longer) was demonstrated to be required between the amino and carboxy termini in order to retain wild-type enzyme activity. The cyclic

Lactam Lactone Lariat Thioester

Fig. 3. Circular ligation mechanism. An expressed fusion protein folds to form an active protein intein. The enzyme catalyzes an N/S acyl shift at the target N-terminal intein junction to produce a thioester, which undergoes transesterification with a side-chain nucle-ophile at the C-terminal intein junction to form a lariat intermediate. Asparagine side-chain cyclization liberates the cyclic product as a lactone, and an X/N acyl shift generates the thermodynamically favored lactam product in vivo

Lactam Lactone Lariat Thioester

Fig. 3. Circular ligation mechanism. An expressed fusion protein folds to form an active protein intein. The enzyme catalyzes an N/S acyl shift at the target N-terminal intein junction to produce a thioester, which undergoes transesterification with a side-chain nucle-ophile at the C-terminal intein junction to form a lariat intermediate. Asparagine side-chain cyclization liberates the cyclic product as a lactone, and an X/N acyl shift generates the thermodynamically favored lactam product in vivo

DHFR produced by SCICLOPPS was shown to be resistant to proteolysis and had steady-state kinetic parameters; and substrate, cofactor, and methotrexate dissociation constants that were indistinguishable from those of the wildtype enzyme at 25 °C. Activity assays conducted after preincubation of wildtype and cyclic DHFR at 65 °C showed an improvement in the thermostability of the cyclic enzyme.

SICLOPPS was also used for the intracellular production of the tyrosinase inhibitor peptide pseudostellarin F (cyclo-SGGYLPPL; Morita et al. 1994). The serine residue in pseudostellarin F served as the nucleophile for the transes-terification portion of the protein ligation mechanism, instead of the cysteine that the wild-type Ssp DnaE C-terminal intein uses. NMR and mass spectroscopy verified the in vivo production of the peptide. Pseudostellarin F produced by SICLOPPS was shown to inhibit recombinant Streptomyces an-tibioticus tyrosinase in vivo demonstrating this integration of intracellular cyclization and functional screening (Scott et al. 1999).

The versatility of the SICLOPPS methodology was demonstrated by its use in the production of combinatorial libraries of small molecules (Scott et al. 2001). Compound libraries are an important tool in the search for inhibitors of ever increasing biological targets and therefore in the battle against disease. Traditionally, organic chemistry has been called upon for the construction of such libraries, thus limiting their size (103-105 members) and ease of construction (Gallop et al. 1994; Gordon et al. 1994; Czarnik 1997); genetic encoding in contrast allows the elaboration of very large libraries (106-109 members) that can be readily interfaced with biological selection. Libraries of cyclic peptides with five randomly variable amino acids and either one or four fixed residues were prepared (Scott et al. 2001). Randomized codons were introduced into the libraries via oligonucleotide synthesis followed by polymerase chain reaction (PCR) cloning, yielding between 107 and 108 transformants. Thus in each individual clone the peptide-encoding region between the intein halves contains a series of five distinct codons that determine the variable region of the cyclic peptide. As the cyclic peptide libraries are biosynthesized within the cell, SICLOPPS can be combined with a functional genetic selection and high-throughput screening to yield a powerful method for identifying potential inhibitors of intracellular targets.

The intracellular production of a library of random cyclic peptides in human cells was recently demonstrated. Retroviral vectors were used in the expression of the DnaB intein from Ssp PCC6803 in the human B cell line BJAB, enabling the function of bacterial split inteins in the cytoplasm of mammalian cells (Kinsella et al. 2002). This has been combined with a functional genetic screen for inhibitors of the interleukin-4 signaling pathway in the BJAB cell line (Schreiber and Crabtree 1992). Disruption of this pathway is a potential therapeutic approach for lowering IgE levels (IL-4 stimulates the germline epsilon gene, a sterile transcript required for IgE isotype class switching) to ameliorate diseases such as allergy and asthma (Oettgen and Geha 2001). Using the genetic screen, 13 cyclic peptides consisting of serine and four random amino acids were isolated. The selective inhibition of germline epsilon transcription by the selected cyclic peptides demonstrated the power and utility of combining random cyclic peptide production with genetic screening.

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