A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability.

Jochen Hecky, Jody M. Mason, Katja M. Arndt, Kristian M. Müller

Research output: Chapter in Book/Report/Conference proceedingOther chapter contribution

7 Citations (Scopus)

Abstract

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such proteins, but the results to date have been of mixed success. Here, we present a robust method involving the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally, selection at physiological temperatures, to generate an enzyme with improved stability. Three cycles of directed evolution comprising of random mutagenesis, DNA shuffling, and selection at 37 degrees C were used, using the bacterial enzyme TEM-1 beta-lactamase as a model protein to yield deletion mutants with in vivo ampicillin resistance levels comparable to wild-type (wt) enzyme. Kinetic studies demonstrate the selected mutant to have a significantly improved thermostability relative to its wt counterpart. Elongation of this mutant to the full-length gene resulted in a beta-lactamase variant with dramatically increased thermostability. This technique was so fruitful that the evolved enzyme retained its maximum catalytic activity even 20 degrees C above its wt parent protein optimum. Thus, structural perturbation by terminal truncation and subsequent compensation by directed evolution at physiological temperatures is a fast, efficient, and highly effective way to improve the thermostability of proteins without the need for selecting at elevated temperatures.

Original languageEnglish
Title of host publicationProtein Engineering Protocols
Subtitle of host publicationPart II
EditorsK. M. Arndt, K. M. Muller
Place of PublicationTotowa, U. S. A.
PublisherHumana Press
Pages275-304
Number of pages30
Volume352
ISBN (Print)9781588290724
DOIs
Publication statusPublished - 13 Feb 2007

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

Fingerprint

Enzyme Stability
Enzymes
Temperature
Mutagenesis
Proteins
DNA Shuffling
Ampicillin Resistance
beta-Lactamases
Genetic Recombination
Peptide Hydrolases
Genes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Hecky, J., Mason, J. M., Arndt, K. M., & Müller, K. M. (2007). A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability. In K. M. Arndt, & K. M. Muller (Eds.), Protein Engineering Protocols: Part II (Vol. 352, pp. 275-304). (Methods in molecular biology (Clifton, N.J.)). Totowa, U. S. A.: Humana Press. https://doi.org/10.1385/1-59745-187-8:275

A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability. / Hecky, Jochen; Mason, Jody M.; Arndt, Katja M.; Müller, Kristian M.

Protein Engineering Protocols: Part II. ed. / K. M. Arndt; K. M. Muller. Vol. 352 Totowa, U. S. A. : Humana Press, 2007. p. 275-304 (Methods in molecular biology (Clifton, N.J.)).

Research output: Chapter in Book/Report/Conference proceedingOther chapter contribution

Hecky, J, Mason, JM, Arndt, KM & Müller, KM 2007, A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability. in KM Arndt & KM Muller (eds), Protein Engineering Protocols: Part II. vol. 352, Methods in molecular biology (Clifton, N.J.), Humana Press, Totowa, U. S. A., pp. 275-304. https://doi.org/10.1385/1-59745-187-8:275
Hecky J, Mason JM, Arndt KM, Müller KM. A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability. In Arndt KM, Muller KM, editors, Protein Engineering Protocols: Part II. Vol. 352. Totowa, U. S. A.: Humana Press. 2007. p. 275-304. (Methods in molecular biology (Clifton, N.J.)). https://doi.org/10.1385/1-59745-187-8:275
Hecky, Jochen ; Mason, Jody M. ; Arndt, Katja M. ; Müller, Kristian M. / A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability. Protein Engineering Protocols: Part II. editor / K. M. Arndt ; K. M. Muller. Vol. 352 Totowa, U. S. A. : Humana Press, 2007. pp. 275-304 (Methods in molecular biology (Clifton, N.J.)).
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