Construction of protein fragment complementation libraries using incremental truncation.

Many proteins can have their peptide backbone cut by proteolytic or genetic means, yet the two fragments can associate to make an active heterodimer. This ‘‘monomer-to-heterodimer conversion’’ is referred to as protein fragment complementation (PFC). Such complementation is the reverse of evolutionary processes in which domains are recruited and fused at the genetic level. Classic examples of protein fragment complementation include ribonuclease S and -galactosidase. Protein fragment complementation can be used to examine theories of protein evolution, protein folding, macromolecular assembly, structure–function relationships, and mapping contacts in membrane proteins. Sites for successful protein bisection for protein fragment complementation are quite varied. Bisection sites need not fall between well-defined domains or structural units and fall within conserved and nonconserved regions, as well as within secondary structure elements such as helixes. Overlapping sequences at the bisections point are often tolerated and, in some cases, even required. For most locations, bisection does not lead to protein fragment complementation, presumably due to inefficient assembly or improper folding of the fragments. For some sites, this can be overcome by fusion of the fragments to dimerization domains to facilitate correct assembly. This is known as assisted protein reassembly (APR). A bisection point for APR