L-Shuffling™

L-Shuffling ™ is a proprietary in vitro "sexual" evolution technology developed by Protéus to allow for poolwise recombination of parental genes in order to overcome the limits of mutagenesis-based approaches.  Pioneering gene shuffling methods were based on PCR-like recombination.

A significant breakthrough has now been achieved with L-Shuffling ™ which involves ligation-based recombination step and therefore permits a high-fidelity recombination of parental genes.

Benefits of L-Shuffling™:

• Allows recombination of both short and long genes
• Works with high and low homology
• Most variants generated using L-Shuffling™ are functional
• Only a limited number of clones needs to be screened for making statistically sure that improved variants will be detected, which is particularly beneficial for complex assays
• Combination of beneficial properties, leading to superior genes

• Combine performances from different parental genes
• Further evolve a protein
• Create new genes with new or superior performances

• Patented technology
• The only custom gene shuffling service
• Evolution of short or long genes, with high or low homology
• No limitation in the number of parental genes
• Generation of mostly functional variants
• Application to all types of proteins (enzymes, therapeutic proteins…)

Please provide:

• Your cDNA(s) cloned in a standard vector. We strongly recommend to send purified plasmids (10 µg in 40 µl TE buffer).
• Corresponding electronic maps and sequences

Protéus can also prepare synthetic genes (wild-type or codon optimised) starting from your electronic sequences. Tags and cleavage sites may be added on your demand to ease purification and downstream processing.

• Shuffled library (as a ligation product in your expression vector)

Phytases are economically significant enzymes that hydolyse phosphoesters of phytic acid and release inorganic phosphate. Phytases are used as feed additives to improve the uptake of phosphate by animals and limit the rejections in environment.

Directed evolution was applied by Protéus to improve the specific activity of a phytase. Starting with a single gene from Bacillus licheniformis, we used EvoSight™ to generate a set of calibrated mutant libraries.

After identification of the optimal library, 5000 variants were screened, of which about two thirds were found to be inactive. 273 mutants showed improved performances when compared to the wild-type enzyme.



Furthermore, EvoSight™ enabled the calculation of the B factor, ie the number of independent beneficial mutations. The value of the B factor was found to be 30.

A high B factor indicates that many different mutations can independently lead to the targeted improvement, and suggests that combining these beneficial mutations iteratively can lead to further improvement.
 
Therefore, the 73 best phytase mutants were selected as parental genes for recombination using L-Shuffling™.

After only 3 L-Shuffling™ rounds, a 50-fold increase in the activity of the phytase was achieved, demonstrating that:

• As predicted by EvoSight™, the protein had a high evolution potential
• The strategy developed thanks to EvoSight™ was successful
• L-Shuffling™ allows enrichment of the library in positive mutations, leading to dramatically improved mutants.