Functionally accepted insertions of proteins within protein domains

Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural dom ain. Dihydrofolate reductase and p-lactamase have been inserted in four dif ferent positions of phosphoglycerate kinase. The resultant chimeric protein s are all overexpressed, and the host as well as the inserted partners are functional, Although not explicitly designed, functional coupling between t he two fused partners was observed in some of the chimeras. These results s how that the tolerance of protein structures to very large structured inser tions is more general than previously expected and supports the idea that t he natural sequence continuity of a structural domain is not required for t he folding process. These results directly suggest a new experimental appro ach to screen, for example, for folded protein in randomized polypeptide se quences.