期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 102, 期 32, 页码 11224-11229出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0502673102
关键词
allostery; beta-lactamase; maltose binding protein
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 beta-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 beta-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 x 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.
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