With the support of our advanced protein engineering platform established for many years, Creative BioMart has developed a variety of scanning mutagenesis methods. Our scientists provide you with a comprehensive customized scanning mutagenesis library construction service.

Scanning mutagenesis is the use of alanine to replace a selected single amino acid residue or any other amino acid, so that all 19 possible substitutions are made at a single amino acid position in the protein. This technique is a valuable tool for systematic probing of protein functions, allowing mapping of protein-protein interaction interfaces, ligand binding sites, and residues that are important for enzyme activity or general function. In addition, it allows protein engineering by incorporating mutations with desired characteristics. Current scanning mutagenesis makes it possible to scan the entire protein sequence of hundreds of amino acids.

Scanning mutagenesis is a powerful protein engineering technique that can study protein functions at a single amino acid resolution and design stable proteins for structural and biophysical work. Generally, alanine scanning mutagenesis is based on PCR amplification of recombinant DNA, followed by enzymatic digestion of the methylated template DNA with the endonuclease DpnI. Although these strategies can provide useful information, it is sometimes necessary to test a wider range of amino acid changes at the target location.

Fig 1. Schematic diagram of codon scanning mutagensis. (Daggett KA, et al., 2009)

Methods of Scanning Mutagenesis

• Two-fragment PCR method: the mutagenic primers in this method are used in two separate PCR reactions, and the resulting fragments are assembled in vitro.
• In vitro transposon mutagenesis method: this method relies on the integration of Mu transposon into target DNA sequence with relatively low site preference. Subsequent removal of the transposon will produce a nucleotide "scar" sequence, leading to protein mutations.
• Pentapeptide scanning mutagenesis: this method is a simple transposon-based program for randomly inserting variable five amino acid boxes into the target protein.

Applications of Scanning Mutagenesis

• Understanding the structure and energy characteristics of hotspots.
• Studying the function of specific amino acid residues in proteins.
• Enzyme activity and function.
• Studying the interaction between proteins.
• Protein binding site.

Service Principle

Creative BioMart is based on innovative, pragmatic and honest, adhering to the tenet of "Quality is our life, providing customers with the best quality service", providing customized services to customers around the world.

We will be glad to discuss details of intended interaction studies with you and develop experimental strategies/methods tailored to your requirement. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, Monday to Friday. Please do not hesitate to contact us for more information or to discuss in detail, if you are interested in our services.

References

1. Forloni M, Liu AY, Wajapeyee N. (2019) Random Scanning Mutagenesis. Cold Spring Harb Protoc. 6.
2. Heydenreich FM, Miljuš T, et al.. (2017) Veprintsev DB. High-throughput mutagenesis using a two-fragment PCR approach. Sci Rep. 7 (1): 6787.
3. Daggett KA, Layer M, Cropp TA. (2009) A general method for scanning unnatural amino acid mutagenesis. ACS Chem Biol. 4 (2): 109-13.

• Homologous Recombination Library Construction Service
• Random and Degenerate Mutagenesis Library Construction Service
• Site-Directed Mutagenesis Library Construction Service