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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

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Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops.

Pascale Mathonet¹, Julie Deherve, Patrice Soumillion

¹Laboratoire de Biochimie Physique et des Biopolymères, Institut des Sciences de la Vie, Université catholique de Louvain, B1348 Louvain-la-Neuve, Belgium.

Protein Science : a Publication of the Protein Society

|September 12, 2006

Summary

Enzyme engineering successfully created new binding sites on TEM-1 beta-lactamase by inserting random peptides into loops. This modification preserved enzyme activity, enabling control via non-natural ligands.

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Area of Science:

Enzymology
Protein Engineering
Biochemistry

Background:

Enzyme activity control is crucial for various applications.
Engineering alternative binding sites on enzymes offers novel control mechanisms.
Loops and turns are natural enzyme substructures suitable for engineering binding sites.

Purpose of the Study:

To genetically engineer alternative binding sites on TEM-1 beta-lactamase by inserting random peptide sequences into specific loops.
To assess the tolerance of these loops to insertions while preserving enzymatic activity.
To identify strategies for increasing the success rate of active mutant generation in enzyme engineering.

Main Methods:

Genetically inserting random peptide sequences into three loops of TEM-1 beta-lactamase.
Assessing mutant activity to determine tolerance to insertions.
Analyzing amino acid distribution in engineered loops.
Comparing tolerance to insertion versus tolerance to mutagenesis.

Main Results:

Insertion tolerance did not correlate with mutagenesis tolerance.
A turn structure tolerated mutagenesis but not insertions.
Rigid loops tolerated insertions with constraints; disulfide bridges were beneficial for some insertions.
Specific loop modifications enhanced the percentage of active mutants.

Conclusions:

Enzyme loops can be engineered to accommodate new binding sites while maintaining activity.
Strategies like disulfide bridge formation and retaining specific residues can improve engineering success.
Combined modifications of multiple loops yielded active hybrid enzymes.