Random deletions from -lactamase yield functional variants

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Sean Devenish, Florian Hollfelder

University of Cambridge, United Kingdom

Enzymes vary widely in size, and there is no clear and direct correlation between size and function. Modern enzymes are likely to carry superfluous residues that are evolutionary artefacts, present as a function of that particular enzyme’s history. Deleting unnecessary residues from enzymes could provide insight into the minimal requirements for function, and provide smaller, more tractable systems for mechanistic studies. Furthermore, smaller proteins would potentially be of interest as efficient modules for synthetic systems. Finally, removing non-critical segments of an enzyme would allow exploration of regions of sequence space that are not readily accessible by the standard methods of random mutagenesis. We describe here a convenient method for carrying out random deletions that we have developed, and its application to the model enzyme -lactamase. The method makes use of an initial transposition step followed by exonuclease digestion to produce deletions of variable length and location. By screening libraries carrying deletions of between 3 and about 40 base pairs for in vivo function, we isolated 24 different functional variants that contained deletions varying from 1-5 codons. The deletions are distributed throughout the enzyme structure and are located largely, but not exclusively, in loops. There is no obvious correlation between the length or location of tolerated deletions and the effect on enzymatic activity.