Eliminating OFF-Frame Clones in Randomized Gene Libraries : An Improved Split β-Lactamase Enrichment System
Abstract
Large, randomized libraries are a key technology for many biotechnological applications. While genetic diversity is the main parameter most libraries direct their resources on, less focus is devoted to ensuring functional IN-frame expression. This study describes a faster and more efficient system based on a split β-lactamase complementation for removal of OFF-frame clones and increase of functional diversity, suitable for construction of randomized libraries. The gene of interest is inserted between two fragments of the β-lactamase gene, conferring resistance to β-lactam drugs only upon expression of an inserted IN-frame gene without stop codons or frameshifts. The preinduction-free system was capable of eliminating OFF-frame clones in starting mixtures of as little as 1% IN-frame clones and enriching to about 70% IN-frame clones, even when their starting rate was as low as 0.001%. The curation system was verified by constructing a single-domain antibody phage display library using trinucleotide phosphoramidites for randomizing a complementary determining region, while eliminating OFF-frame clones and maximizing functional diversity.
Info
Journal Article, 2023
UN SDG Classification
DK Main Research Area
Science/Technology
