Saturation mutagenesis

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Saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants at position 5 are shown below. Site saturation mutagenesis.svg
Saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants at position 5 are shown below.

Saturation mutagenesis, or site saturation mutagenesis (SSM), or simply site saturation, is a random mutagenesis technique used in protein engineering, in which a single codon or set of codons is substituted with all possible amino acids at the position. [1] There are many variants of the site saturation technique, from paired site saturation (saturating two positions in every mutant in the library) to scanning single-site saturation (performing a site saturation at each site in the protein, resulting in a library of size 20n, where n is the number of peptides in the protein, or n-site saturation, where n sites in a peptide would be site saturated, with a library size of 20n, where if the length of your peptide is n, you have complete randomization.

Contents

Method

Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red). Many such primers containing degeneracy in the non-complementary region are pooled into the same PCR, resulting in many different PCR products with different mutations in that region (individual mutants shown with different colors below). Site-directed mutagenesis library cloning steps.pdf
Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red). Many such primers containing degeneracy in the non-complementary region are pooled into the same PCR, resulting in many different PCR products with different mutations in that region (individual mutants shown with different colors below).

Saturation mutagenesis is commonly achieved by site-directed mutagenesis PCR with a randomised codon in the primers (e.g. SeSaM) [2] or by artificial gene synthesis, with a mixture of synthesis nucleotides used at the codons to be randomised. [3]

Different degenerate codons can be used to encode sets of amino acids. [1] Because some amino acids are encoded by more codons than others, the exact ratio of amino acids cannot be equal. Additionally, it is usual to use degenerate codons that minimise stop codons (which are generally not desired). Consequently, the fully randomised 'NNN' is not ideal, and alternative, more restricted degenerate codons are used. 'NNK' and 'NNS' have the benefit of encoding all 20 amino acids, but still encode a stop codon 3% of the time. Alternative codons such as 'NDT', 'DBK' avoid stop codons entirely, and encode a minimal set of amino acids that still encompass all the main biophysical types (anionic, cationic, aliphatic hydrophobic, aromatic hydrophobic, hydrophilic, small). [1] In the case there is no restriction to use a single degenerate codon only, it is possible to reduce the bias considerably. [4] [5] Several computational tools were developed to allow high level of control over the degenerate codons and their corresponding amino acids. [6] [7] [8]

Degenerate codon No. of codonsNo. of amino acidsNo. of stopsAmino acids encoded
NNN64203All 20
NNK / NNS32201All 20
NDT12120RNDCGHILFSYV
DBK18120ARCGILMFSTWV
NRT880RNDCGHSY

Applications

Saturation mutagenesis is commonly used to generate variants for directed evolution. [9] [10]

See also

References

  1. 1 2 3 Reetz, M. T.; Carballeira J. D. (2007). "Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes". Nature Protocols. 2 (4): 891–903. doi:10.1038/nprot.2007.72. PMID   17446890. S2CID   37361631.
  2. ↑ Zheng, Lei; Baumann, Ulrich; Reymond, Jean-Louis (2004-07-15). "An efficient one-step site-directed and site-saturation mutagenesis protocol". Nucleic Acids Research. 32 (14): e115. doi:10.1093/nar/gnh110. ISSN   0305-1048. PMC   514394 . PMID   15304544.
  3. ↑ Reetz, Manfred T.; Prasad, Shreenath; Carballeira, José D.; Gumulya, Yosephine; Bocola, Marco (2010-07-07). "Iterative Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods". Journal of the American Chemical Society. 132 (26): 9144–9152. doi:10.1021/ja1030479. ISSN   0002-7863. PMID   20536132.
  4. ↑ Kille, Sabrina; Acevedo-Rocha, Carlos G.; Parra, Loreto P.; Zhang, Zhi-Gang; Opperman, Diederik J.; Reetz, Manfred T.; Acevedo, Juan Pablo (2013-02-15). "Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis". ACS Synthetic Biology. 2 (2): 83–92. doi:10.1021/sb300037w. PMID   23656371.
  5. ↑ Tang, Lixia; Wang, Xiong; Ru, Beibei; Sun, Hengfei; Huang, Jian; Gao, Hui (June 2014). "MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites". BioTechniques. 56 (6): 301–302, 304, 306–308, passim. doi: 10.2144/000114177 . ISSN   1940-9818. PMID   24924390.
  6. ↑ Halweg-Edwards, Andrea L.; Pines, Gur; Winkler, James D.; Pines, Assaf; Gill, Ryan T. (September 16, 2016). "A Web Interface for Codon Compression". ACS Synthetic Biology. 5 (9): 1021–1023. doi:10.1021/acssynbio.6b00026. ISSN   2161-5063. PMID   27169595.
  7. ↑ Engqvist, Martin K. M.; Nielsen, Jens (2015-04-30). "ANT: Software for Generating and Evaluating Degenerate Codons for Natural and Expanded Genetic Codes". ACS Synthetic Biology. 4 (8): 935–938. doi:10.1021/acssynbio.5b00018. PMID   25901796.
  8. ↑ Kell, Douglas B.; Day, Philip J.; Breitling, Rainer; Green, Lucy; Currin, Andrew; Swainston, Neil (2017-07-10). "CodonGenie: optimised ambiguous codon design tools". PeerJ Computer Science. 3: e120. doi: 10.7717/peerj-cs.120 . ISSN   2376-5992.
  9. ↑ Chica, Robert A.; et al. (2005). "Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design". Current Opinion in Biotechnology. 16 (4): 378–384. doi:10.1016/j.copbio.2005.06.004. PMID   15994074.
  10. ↑ Shivange, Amol V; Marienhagen, Jan; Mundhada, Hemanshu; Schenk, Alexander; Schwaneberg, Ulrich (2009-02-01). "Advances in generating functional diversity for directed protein evolution". Current Opinion in Chemical Biology. Biocatalysis and Biotransformation/Bioinorganic Chemistry. 13 (1): 19–25. doi:10.1016/j.cbpa.2009.01.019. PMID   19261539.
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