Converting one enzyme into another is challenging due to the uneven distribution
of important amino acids for function in both protein sequence and structure. We
report a strategy for protein engineering allowing an organized mixing and
matching of genetic material that leverages lower throughput with increased
quality of screens. Our approach successfully tested the contribution of each
surface-exposed loop in the trypsin fold alone and the cooperativity of their
combinations towards building the substrate selectivity and Na(+)-dependent
allosteric activation of the protease domain of human coagulation factor Xa into
a bacterial trypsin. As the created proteases lack additional protein domains
and protein co-factor activation mechanism requisite for the complexity of blood
coagulation, they are stepping-stones towards further understanding and
engineering of artificial clotting factors.
