Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis

Cheng, Feng; Xu, Jian-Zhong; Xiang, Chao; Liu, Zhi‐Qiang; Zhao, Lingyun

doi:10.1007/s10529-016-2278-x

Cited by 19 publications

(13 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Site saturation mutagenesis is a technique, whereby a set of codons is randomized to produce a library of variants with every possible amino acid at each randomized position in the target region. [17][18][19] This method can be contrasted with error-prone PCR, in which many mutations that do not correspond to amino acid substitution are generated. Recently, it has become possible to create site saturation mutagenesis libraries using commercially available custom oligonucleotide libraries.…”

Section: Generation Of Thermostable Moloney Murine Leukemia Virus Reverse Transcriptase Variants Using Site Saturation Mutagenesis Librarmentioning

confidence: 99%

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system

Katano

Baba

et al. 2017

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

show abstract

Section: Generation Of Thermostable Moloney Murine Leukemia Virus Reverse Transcriptase Variants Using Site Saturation Mutagenesis Librarmentioning

confidence: 99%

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system

Katano

Baba

et al. 2017

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

show abstract

“…Method is robust and reliable, though cumbersome for multiple mutation sites. >90% 100% ( An et al, 2005 ; Bryksin and Matsumura, 2010 ; Cheng et al, 2017 ; Heckman and Pease, 2007 ; Hussain and Chong, 2016 ; Wäneskog and Bjerling, 2014 ; Wei et al, 2012 ; Williams et al, 2014 ; Xiao and Pei, 2011 ) Asymmetric PCR A single-stranded gene fragment is amplified by asymmetric PCR using mutagenic primers, which is then used as a megaprimer to introduce mutations into full sequence. Method is robust and reliable, though cumbersome for multiple mutation sites.…”

Section: Targeted Diversity Creationmentioning

confidence: 99%

“…5 A). Further developments in this approach have made it amenable to combinatorial mutations ( An et al, 2005 ; Cheng et al, 2017 ; Wäneskog and Bjerling, 2014 ; Wei et al, 2012 ). Similarly, mutagenesis using asymmetric PCR employs two steps, where mutagenic primers are used to create single-stranded products, which are then deployed as megaprimers to introduce these mutations in the second step ( Bi et al, 2012 ; Sadler et al, 2018 ).…”

Section: Targeted Diversity Creationmentioning

confidence: 99%

The evolving art of creating genetic diversity: From directed evolution to synthetic biology

Currin

Parker

Robinson

et al. 2021

Biotechnology Advances

View full text Add to dashboard Cite

The ability to engineer biological systems, whether to introduce novel functionality or improved performance, is a cornerstone of biotechnology and synthetic biology. Typically, this requires the generation of genetic diversity to explore variations in phenotype, a process that can be performed at many levels, from single molecule targets ( i.e. , in directed evolution of enzymes) to whole organisms ( e.g. , in chassis engineering). Recent advances in DNA synthesis technology and automation have enhanced our ability to create variant libraries with greater control and throughput. This review highlights the latest developments in approaches to create such a hierarchy of diversity from the enzyme level to entire pathways in vitro , with a focus on the creation of combinatorial libraries that are required to navigate a target's vast design space successfully to uncover significant improvements in function.

show abstract

“…Selected residues can be combined iteratively or simultaneously . Numerous multiple site saturation mutagenesis (MSSM) methods have been published [e.g., simple‐MSSM, PFunkel, multiple patch cloning (MUPAC), OmniChange, combinatorial active site saturation test (CAST)] and are limited in the number of simultaneously mutated positions…”

Section: Introductionmentioning

confidence: 99%

A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis

et al. 2018

View full text Add to dashboard Cite

Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (20 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity.

show abstract

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis

Abstract: The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Cited by 19 publications

References 19 publications

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system

The evolving art of creating genetic diversity: From directed evolution to synthetic biology

A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis

Contact Info

Product

Resources

About