Protein Mutagenesis Library Construction

Creative Biolabs offers customers protein mutagenesis library construction services with a complexity of up to 10⁹. These libraries will be delivered as dsDNA, clones in your plasmid vectors of choice, or specified transformants in glycerol stocks.

Standard oligonucleotide-directed site-specific mutagenesis methods can produce well-defined sequence changes, but the creation of large numbers of mutants can be both time- and cost-prohibitive. In contrast, the mutagenesis oligo libraries that can be created give you precise, codon-level control over the variants that are produced. Use the oligo libraries together with the oligo library mutagenesis system reagent kit to quickly create large libraries of well-defined mutants that are ideally suited for applications such as scanning mutagenesis and mutagenesis that is targeted to many specific amino acids of interest.

Protein mutagenesis libraries have been widely used to optimize protein functions by identifying and modifying critical residues in order to alter the protein structurally and functionally. It is an important method of protein engineering for generating novel proteins with increased solubility, stability, expression levels and so on. In general, protein mutagenesis libraries are collections of DNA oligonucleotides (oligoes) that can be used for the creation of families of variant genes and proteins for functional studies. We have extensive experiences in the field of mutagenesis library construction of the following types:

Single-Site Mutagenesis Libraries
Scanning Point Mutation Libraries
Randomized and Degenerated Libraries

Figure 1. The general scheme of site saturation mutagenesis approach for the mutagenesis library construction. (Francesca Valetti et al., 2013)

These mutant libraries enable the systematic study of protein properties, regulation, and function. However, the trend for enzyme optimization has been moving from pure “blind” to “semi-rational” approaches in order to accommodate the demands of rapid creation of novel industrial biocatalysts. Mutagenesis of “hot spots” and regions are generally guided by mechanistic and structural information, and also aided by computational methods like B-FITTER, PatchFinder, and Rate4Site. The option of generating small focused mutant libraries at the defined positions has been more preferred.

In designing the libraries, we will also look into the redundancy of the degenerated codon, so as to keep a high quality of the constructed library and screen low frequency positive colonies more easily.

Error-prone PCR has been shown to produce a broader non-Poisson distribution of mutations, which is consistent with a detailed model of PCR. As error rates increase, this distribution leads directly to the observed excesses in functional clones. Very low mutation rates result in many functional sequences, but only a small number are unique. By contrast, very high mutation rates produce mostly unique sequences, but few retain function. We can decide an optimal mutation rate that balances uniqueness and retention of function, based on your protein and the mutagenesis protocol.

Reference

Francesca Valetti and Gianfranco Gilardi. Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis. Biomolecules 2013, 3, 778-811; doi:10.3390/biom3040778

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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