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, [1999], Proc. Natl. Acad. Sci. USA 96, 3562-3567). This heterodimer (B13) 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, B13. The activity of B13 is comparable to that of the wild type and the pH-dependent kinetics of B13 turned out to be nearly identical to those of the wild type, indicating that B13 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.
Copyright 2000 Academic Press.