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PEDS Advance Access published online on March 15, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi005
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received January 27, 2005
Accepted February 1, 2005

Article

Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state

C. Roodveldt 1 and D. S. Tawfik 1*

1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

* To whom correspondence should be addressed.
D. S. Tawfik, E-mail: tawfik{at}weizmann.ac.il


  Abstract

Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another--and often overlooked--possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.

Keywords: apoenzyme; directed evolution; Escherichia coli; heterologous expression; organophosphorus hydrolase; phosphotriesterase; promiscuity; recombinant.
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