The unexpected catalytic properties of a heterodimer of GAR transformylase

We have developed an efficient expression and purifcation protocol for a he terodimer of glycinamide ribonucleotide transformylase that was identified in incremental truncation libraries, a general combinatorial method for pro tein fragment complementation (M. Ostermeier, A. E. Nixon, J. H. Shim, and S. J. Benkovic, [1999], Proc. Natl. Acad. Sci. USA 96, 3562-3567). This het erodimer (B13) containing both a bisection point and a deletion in conserve d residues close to the active site was expressed and purified in high yiel d using Intein methodology. The N-terminus fragment(l-lll) and C-terminus f ragment (M114-212) were also expressed separately as stable proteins. When these two fragments were mixed together, they associate at a highly specifi c 1:1 ratio to give only the active heterodimer, B13. The activity of B13 i s 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 tha t B13 operates in the same mechanism as the wild type. This result demonstr ated that cutting within conserved regions is a viable domain separation an d confirmed the generality of using incremental truncation for protein frag ment complementation. (C) 2000 Academic Press.