Directed evolution of protein switches and their application to the creation of ligand-binding proteins
AUTOR(ES)
Guntas, Gurkan
FONTE
National Academy of Sciences
RESUMO
We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 β-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP–BLA hybrids in which maltose is a positive or negative effector of β-lactam hydrolysis. Some of these MBP–BLA switches were effectively “on–off” in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth/no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 × 106 variants, MBP–BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a “sucrose-binding protein,” illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1183557Documentos Relacionados
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