Protein switches created by non-homologous recombination

Gurkan Guntas, Jing Liang, Jin Ryoun Kim, Thomas J. Mansell, Jason Boock, Marc Ostermeier

Research output: Contribution to conferencePaper

Abstract

We have developed a directed evolution approach for creating protein switches involving the in vitro recombination of two non-homologous genes with the prerequisite input and output functions. We have recombined the genes coding for TEM1 b-lactamase (BLA) and the E. coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is either a positive or negative effector of b-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. One MBP-BLA switch was identified that could be positively allosterically regulated by maltose and negatively allosterically regulated by Zn2+, which is surprising considering that neither BLA nor MBP bind Zn2+. The origin of this effect has implications for how novel protein function evolves. The ability of the MBP-BLA 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 wildtype MBP converted MBP into a 'sucrose-binding protein,' illustrating the switches potential as a tool to rapidly identify novel ligand-binding proteins.

Original languageEnglish (US)
Number of pages1
StatePublished - 2005
Event05AIChE: 2005 AIChE Annual Meeting and Fall Showcase - Cincinnati, OH, United States
Duration: Oct 30 2005Nov 4 2005

Other

Other05AIChE: 2005 AIChE Annual Meeting and Fall Showcase
CountryUnited States
CityCincinnati, OH
Period10/30/0511/4/05

ASJC Scopus subject areas

  • Engineering(all)

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    Guntas, G., Liang, J., Kim, J. R., Mansell, T. J., Boock, J., & Ostermeier, M. (2005). Protein switches created by non-homologous recombination. Paper presented at 05AIChE: 2005 AIChE Annual Meeting and Fall Showcase, Cincinnati, OH, United States.