We have developed an efficient expression and purification protocol for a heterodimer of glycinamide ribonucleotide transformylase that was identified in incremental truncation libraries, a general combinatorial method for protein fragment complementation (M. Ostermeier, A. E. Nixon, J. H. Shim, and S. J. Benkovic, , Proc. Natl. Acad. Sci. USA 96, 3562-3567). This heterodimer (B 13) containing both a bisection point and a deletion in conserved residues close to the active site was expressed and purified in high yield using Intein methodology. The N-terminus fragment (1-111) and C-terminus fragment (M114-212) were also expressed separately as stable proteins. When these two fragments were mixed together, they associate at a highly specific 1:1 ratio to give only the active heterodimer, B 13. The activity of B 13 is comparable to that of the wild type and the pH-dependent kinetics of B 13 turned out to be nearly identical to those of the wild type, indicating that B 13 operates in the same mechanism as the wild type. This result demonstrated that cutting within conserved regions is a viable domain separation and confirmed the generality of using incremental truncation for protein fragment complementation.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Drug Discovery
- Organic Chemistry