GeneArt Site-Saturation Mutagenesis

Site-saturated mutagenesis is a technique used to generate a library of mutant proteins by systematically replacing a wild-type amino acid in a functionally important location of the protein with each alternative amino acid. Also known as sequential permutation, site-saturation mutagenesis in combination with a powerful screening assay is a systematic mutation strategy to identify amino acid substitutions that fulfill your protein engineering goals.

The difference between site-directed mutagenesis and site-saturation mutagenesis is that site-directed mutagenesis introduces specific, targeted mutations at a particular site to study precise alterations, whereas site saturation mutagenesis creates a library of all possible mutations at a specific site to explore the full range of functional effects.

Once you’ve identified variants with beneficial substitutions, the GeneArt combinatorial library service can be used to create material for more targeted directed evolution experiments to further optimize your protein.

Why choose GeneArt site-saturation mutagenesis service?

Systematic identification of beneficial amino acid substitutions—check every possible variant at each position
Structural information about the protein is not needed, but it can be beneficial
Fewer variants to screen—no oversampling required when all clones are delivered in separate tubes
Fast delivery times—get your project moving quickly
Cost-efficient

Applications of site-saturation mutagenesis

Identify beneficial or detrimental amino acid substitutions
Help increase affinity, specificity, activity, heat stability, detergent tolerance, and other features
Change substrate specificity or enantioselectivity
Help reduce homology between two related proteins (avoid IP issues)
Identify active sites or receptor-binding sites

Quality control

Constructs supplied individually are 100% sequence-verified
Bulk sequencing of pooled constructs to verify that the nucleotide content at the requested positions is degenerate and the unmutated regions are intact

Site-saturation mutagenesis services: Selection guide

We offer several sequential permutation options to fit a wide array of research needs. You can create variants at one or more codons and choose to receive individual or pooled constructs. When evaluating most of the possible 19 non-wild type amino acids is sufficient, we offer the GeneArt site-saturation mutagenesis average 16 and GeneArt site-saturation mutagenesis minimum 16 services that are delivered as individual clones encoding an average of 16 or a minimum of 16 substitute amino acids at each position, respectively.

Variant constructs are subcloned into the vector of your choice.

GeneArt site-saturation mutagenesis pool of one position Figure 1. GeneArt site-saturation mutagenesis pool of one position—Pooled variant generation at a single codon site.	A mixture of all 19 non-wild type variants at one codon position Supplied as a pooled glycerol stock
GeneArt site-saturation mutagenesis pool of all positions Figure 2. GeneArt site-saturation mutagenesis pool of all positions—Pooled variant library across multiple codon sites.	A mixture of all 19 non-wild type variants at more than one codon position Supplied as a pooled glycerol stock
GeneArt site-saturation mutagenesis average 16 Figure 3. GeneArt site-saturation mutagenesis average 16—Individual clone variants with an average of 16 substitutions per position.	An average of 16 different non-wild type amino acids per position Supplied as individual glycerol stocks
GeneArt site-saturation mutagenesis minimum 16 Figure 4. GeneArt site-saturation mutagenesis minimum 16—Individual clone variants with a minimum of 16 substitutions per position.	At least 16 different non-wild type amino acids per position Supplied as individual glycerol stocks
GeneArt site-saturation mutagenesis 19 Figure 5. GeneArt site-saturation mutagenesis 19—Individual clone variants representing all 19 amino acid substitutions per site.	All 19 different non-wild type amino acids per position Supplied as individual glycerol stocks

Get started with your site-saturation mutagenesis project

Please download the GeneArt site-saturation mutagenesis custom service requirements form to submit project information.

For secure data transfer please use the GeneArt Services Dashboard (login required).

For further information regarding this service or the order process, please contact geneartsupport@thermofisher.com.

Case study (customer example): Functional mapping of RNA polymerase with site-saturation mutagenesis

Analysis of bridge helix region of RNA-polymerase subunit mjA´ of Methanocaldococcus jannaschii

To determine the impact of structural changes on RNA polymerase enzyme activity Tan et al. characterized the functional properties of mjA´ subunit mutants. A subset of mutants was produced by GeneArt site-saturation mutagenesis services. Activity testing of the resulting single-point mutants demonstrated a wide spectrum of different phenotypes (Figure 6), including some hyperactive variants having higher activity than wild type. These were subjected to further investigations.

More details can be found in the original article, which can be accessed here.