geneart-site-saturation-mutagenesis-service

Systematic Mutagenesis

Site-saturation mutagenesis is known to systematically replace wild type amino acid coding sequences with sequences encoding all 19 non–wild type amino acids at the position(s) you specify. Also known as sequential permutation, site-saturation mutagenesis in combination with a powerful screening assay is the most systematic mutation strategy to identify amino acid substitutions that fulfill your protein engineering goals.

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.

Get Your Project Started

Please download the GeneArt Site-Saturation Mutagenesis Custom Service Requirements Form  to submit project information. For secure data transfer please register at our customer portal.

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

Case study: Functional Mapping of RNA Polymerase (Customer Example)

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 were produced by GeneArt site-saturation mutagenesis services.

Activity testing of the resulting single-point mutants demonstrated a wide spectrum of different phenotypes (see graphic below), 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.

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Options for Site-Saturation Mutagenesis

We offer several options for sequential permutation to fit a wide array of research needs. You can choose to create variants at one or more codons, and receive constructs individually or pooled. 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 provide 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
  • 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
  • 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
  • An average of 16 different non–wild type amino acids per position
  • Supplied as individual glycerol stocks
GeneArt™ Site-Saturation Mutagenesis Minimum 16
  • At least 16 different non–wild type amino acids per position
  • Supplied as individual glycerol stocks
GeneArt™ Site-Saturation Mutagenesis 19
  • All 19 different non–wild type amino acids per position
  • Supplied as individual glycerol stocks
  • 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
  • Identify beneficial or detrimental amino acid substitutions
  • Increase affinity, specificity, activity, heat stability, detergent tolerance, etc.
  • Change substrate specificity or enantioselectivity
  • Reduce homology between two related proteins (avoid IP issues)
  • Identify active sites or receptor-binding sites
  • 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 is required when all clones are delivered in separate tubes
  • Fast delivery times—get your project moving quickly
  • Extremely cost-efficient
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
Applications
  • Identify beneficial or detrimental amino acid substitutions
  • Increase affinity, specificity, activity, heat stability, detergent tolerance, etc.
  • Change substrate specificity or enantioselectivity
  • Reduce homology between two related proteins (avoid IP issues)
  • Identify active sites or receptor-binding sites
Advantages
  • 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 is required when all clones are delivered in separate tubes
  • Fast delivery times—get your project moving quickly
  • Extremely cost-efficient

References

Literature

Bridge helix and trigger loop perturbations generate superactive RNA polymerases

FAQs

Stylesheet for Classic Wide Template adjustments