br Experimental methods br Results and discussion br Conclus
Results and discussion
Conclusions Here we describe the first reported method for high-yield 4-P-PDOT of active and soluble mature CPG2 (in the absence of the leader peptide) and its individual catalytic and dimerization domains in E. coli. Such a method is of key importance for future structural and binding studies involving this enzyme, which we suggest may have been hampered by difficulties in obtaining large yields of the protein as well as labelling of the protein. Using the condensation methods outlined by Sivashanmugan et al.  and Marley et al.30, we can routinely produce milligrams quantities of 15N and 2H/13C/15N isotopically-labelled protein suitable for NMR studies. Solution NMR is well-suited to the investigation of ligand-binding characteristics of CPG2, which is of high importance in applications using this enzyme (such as ADEPT). NMR can also be used to obtain information on conformational dynamics, which may have held back crystallographic approaches to ligand-binding studies thus far. Although the NMR data described here for the full-length, mature CPG2 is preliminary, and included here only to illustrate the improvement in peak width as enzyme size was decreased, work is ongoing to use our optimised expression protocol to identify conditions that yield a full complement of 3D NMR data to facilitate assignment. We utilized a “divide-and-conquer” approach to investigate, for the first time, the isolated catalytic and dimerization domains in CPG2. This was motivated in part by the quest for high-quality NMR peak shapes, and in part as an effort to reduce the immunogenicity of CPG2 by reducing the overall size while maintaining activity. NMR data clearly indicated that the isolated dimerization domain did not fold, but the isolated catalytic domain (created by fusing the two non-contiguous regions of this domain through a single Ala residue, Fig. 6A) was able to fold in the absence of the dimerization domain and cleave MTX with an activity 62% that of wild-type levels. To further narrow down the components necessary for activity, the largest contiguous region of the catalytic domain (CPG2CAT′, Fig. 6A) was expressed and purified. The CPG2CAT′ protein appeared folded from the appearance of the NMR data (i.e. peaks are narrow and well-dispersed), but did not retain the parent protein’s enzymatic activity. This lack of activity demonstrates that, while the dimerization domain is not required, the C-terminal region of the catalytic domain (residues 323–415) is critical in substrate recognition/activity. The residues proposed to form the active site in CPG2  are mapped onto the crystal structure in Fig. 7 and illustrate that the two non-contiguous regions come together to form the complete active site. Removal of the C-terminal region leads to removal of two residues from the active site (shown in dark grey), specifically His 385 and Arg 324, the later of which is the only residue to have been shown experimentally via mutagenesis to play a role in catalysis. Therefore future work will focus on NMR structural elucidation and ligand binding studies for the CPG2CAT protein, which shows great promise for downstream structural studies, as well as investigation of the immunogenicity of the CPG2CAT protein.
Acknowledgements This work was supported by a Biotechnology and Biological Sciences Research Council Industrial Case Studentship (BB/I015965/1) awarded to AMD and PD. The authors wish to thank M. Chow for helpful discussions, I. Prokes (University of Warwick, Coventry, UK) for NMR assistance, and N. Chmel (University of Warwick, Coventry, UK) for circular dichroism assistance.
Introduction The US Food and Drug Administration has approved >180 therapeutic peptides and proteins for many applications and disease treatments. Because most of these proteins and peptides are smaller in size than the kidney filtration cutoff of ~70 kDa, they do not have optimal pharmacokinetics. Thus, these protein and peptide therapeutics have short half-lives in vivo, due to the action of proteases and the generation of antibodies against them.(Werle and Bernkop-Schnurch, 2006)