Biosynthesis of a Head-to-Tail Cyclized Protein with Improved Biological Activity

Head-to-tail (backbone) peptide cyclization involves condensing the Nand C-termini of a peptide together and is typically used to rigidify structure and improve in ViVo stability.1 Although backbone cyclization is routinely used to modify the properties of small bioactive peptides as part of drug discovery programs, its application to proteins remains largely unexplored.2 This is primarily because the synthetic methodologies developed for the cyclization of small peptides cannot be easily extended to much larger protein systems. Nonetheless, protein cyclization remains of considerable interest to the protein engineering and protein folding communities3 since it is expected to provide useful insights into the importance of the Cand N-termini of a protein in folding, structural stability, and, consequently, biological activity. In the present work, we describe a novel biosynthetic process, based on an intramolecular chemical ligation reaction, which allows the cyclization of recombinant polypeptide precursors. This approach has been successfully applied to the generation of a circular version of a Src homology 3 (SH3) domain with improved biological activity over the wild-type protein. Several strategies for the preparation of circular polypeptides from unprotected linear precursors have been described. In pioneering studies in this area, Creighton and co-workers used a chemical cross-linking approach to prepare a backbone cyclized version of bovine pancreatic trypsin inhibitor.2a More recently, chemical2b,c,4 and enzymatic2d intramolecular ligation methods have been developed which allow linear synthetic peptides to be efficiently cyclized under aqueous conditions. However, the requirement for synthetic peptide precursors has limited these chemical/enzymatic cyclization approaches to relatively small polypeptide systems. One potential solution to this size problem would be to use recombinant polypeptides as precursors, thereby allowing large polypeptides and even proteins to be backbonecyclized. In principle, it should be possible to generate a circular recombinant protein by using an intramolecular version of the native chemical ligation approach.5 Native chemical ligation involves the chemoselective reaction that occurs between an N-terminal cysteine residue in one peptide and an R-thioester group within a second peptide, resulting in the formation of a normal peptide bond. Importantly, incorporation of both of these reactive moieties within the same synthetic polypeptide leads to efficient backbone cyclization.2b,c,4 Considerable progress has been made in generating recombinant polypeptides for use in native chemical ligation reactions: Verdine and co-workers have used a mutagenesis/factor Xa proteolysis strategy to generate reactive recombinant N-terminal cysteine proteins,6 while we and others have shown that recombinant polypeptide R-thioesters can be prepared by chemically intercepting a naturally occurring proteinsplicing reaction.7 To date, recombinant polypeptide segments have been used only in intermolecular native chemical ligation reactions.7 Here we describe a straighforward route to backbonecyclized recombinant polypeptides based on an intramolecular version of native chemical ligation (Figure 1). As a demonstration of our biosynthetic cyclization strategy, we attempted to cyclize the N-terminal SH3 domain from the c-Crk adaptor protein.8 This 57-residue protein domain possesses a globular structure9 composed of five !-strands which position the Nand C-termini in close proximity.10 A bacterial expression plasmid was prepared in which the gene corresponding to the SH3 domain (residues Y136 to Y190 of murine c-Crk) was cloned into a commercially available intein expression system.11 The SH3 domain was modified at the DNA level to append the sequence MIEGRC at the N-terminus and to add an extra Gly residue at the C-terminus. The MIEGRC motif contains a factor Xa proteolysis site and allows the generation of an N-terminal Cys upon in Vitro proteolysis.6,7e Importantly, this motif acts as a cysteine-protecting group, preventing premature ligation reactions.7e The addition of the C-terminal Gly residue was carried out to improve the kinetics of cyclization as well as to stabilize the formation of the new loop between the Nand C-termini.12 The recombinant fusion protein (1) was expressed in Escherichia coli to give, after affinity purification, a major component on SDS-PAGE with the correct apparent molecular weight (Figure 2A). A secondary band representing less than 10% of total protein was also detected. This was assigned as the intein-CBD protein fragment and is presumably due to some premature cleavage of the fusion protein (1). As illustrated in Figure 1, cyclization of the recombinant SH3 domain was achieved by simply treating the fusion protein (1) adsorbed on chitin beads with factor Xa protease at pH 7.2 for 10 h. This proteolysis step afforded N-terminal Cys fusion protein (2) which spontaneously reacted in an intramolecular fashion to yield the corresponding circular SH3 domain (3) with concomitant cleavage from the beads. As shown in Figure 2A and B, the cyclization process was extremely clean, allowing the circular SH3 domain (3) to be readily purified