Cargando…
Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy
Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PC...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2018
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013526/ https://www.ncbi.nlm.nih.gov/pubmed/29785500 http://dx.doi.org/10.1007/s00253-018-9041-2 |
_version_ | 1783334031325134848 |
---|---|
author | Li, Aitao Acevedo-Rocha, Carlos G. Reetz, Manfred T. |
author_facet | Li, Aitao Acevedo-Rocha, Carlos G. Reetz, Manfred T. |
author_sort | Li, Aitao |
collection | PubMed |
description | Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PCR-based approach using NNK codon degeneracy. However, in the case of difficult-to-randomize genes, SSM may not deliver all of the expected 19 mutants, which compels the user to invest further efforts by applying site-directed mutagenesis for the construction of the missing mutants. To solve this problem, we developed a two-step PCR-based technique in which a mutagenic primer and a non-mutagenic (silent) primer are used to generate a short DNA fragment, which is recovered and then employed as a megaprimer to amplify the whole plasmid. The present two-step and older one-step (partially overlapped primer approach) procedures were compared by utilizing cytochrome P450-BM3, which is a “difficult-to-randomize” gene. The results document the distinct superiority of the new method by checking the library quality on DNA level based on massive sequence data, but also at amino acid level. Various future applications in biotechnology can be expected, including the utilization when constructing mutability landscapes, which provide semi-rational information for identifying hot spots for protein engineering and directed evolution. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00253-018-9041-2) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6013526 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-60135262018-06-25 Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy Li, Aitao Acevedo-Rocha, Carlos G. Reetz, Manfred T. Appl Microbiol Biotechnol Applied Genetics and Molecular Biotechnology Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PCR-based approach using NNK codon degeneracy. However, in the case of difficult-to-randomize genes, SSM may not deliver all of the expected 19 mutants, which compels the user to invest further efforts by applying site-directed mutagenesis for the construction of the missing mutants. To solve this problem, we developed a two-step PCR-based technique in which a mutagenic primer and a non-mutagenic (silent) primer are used to generate a short DNA fragment, which is recovered and then employed as a megaprimer to amplify the whole plasmid. The present two-step and older one-step (partially overlapped primer approach) procedures were compared by utilizing cytochrome P450-BM3, which is a “difficult-to-randomize” gene. The results document the distinct superiority of the new method by checking the library quality on DNA level based on massive sequence data, but also at amino acid level. Various future applications in biotechnology can be expected, including the utilization when constructing mutability landscapes, which provide semi-rational information for identifying hot spots for protein engineering and directed evolution. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00253-018-9041-2) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2018-05-21 2018 /pmc/articles/PMC6013526/ /pubmed/29785500 http://dx.doi.org/10.1007/s00253-018-9041-2 Text en © The Author(s) 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Applied Genetics and Molecular Biotechnology Li, Aitao Acevedo-Rocha, Carlos G. Reetz, Manfred T. Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy |
title | Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy |
title_full | Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy |
title_fullStr | Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy |
title_full_unstemmed | Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy |
title_short | Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy |
title_sort | boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step pcr strategy |
topic | Applied Genetics and Molecular Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013526/ https://www.ncbi.nlm.nih.gov/pubmed/29785500 http://dx.doi.org/10.1007/s00253-018-9041-2 |
work_keys_str_mv | AT liaitao boostingtheefficiencyofsitesaturationmutagenesisforadifficulttorandomizegenebyatwosteppcrstrategy AT acevedorochacarlosg boostingtheefficiencyofsitesaturationmutagenesisforadifficulttorandomizegenebyatwosteppcrstrategy AT reetzmanfredt boostingtheefficiencyofsitesaturationmutagenesisforadifficulttorandomizegenebyatwosteppcrstrategy |