简单MSSM：一种用于同时多位点饱和诱变的简单高效方法。

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

作者信息

Cheng Feng, Xu Jian-Miao, Xiang Chao, Liu Zhi-Qiang, Zhao Li-Qing, Zheng Yu-Guo

机构信息

Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People's Republic of China.

College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, People's Republic of China.

出版信息

Biotechnol Lett. 2017 Apr;39(4):567-575. doi: 10.1007/s10529-016-2278-x. Epub 2017 Jan 2.

DOI:10.1007/s10529-016-2278-x

PMID:28044225

Abstract

OBJECTIVE

To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation.

RESULTS

A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G).

CONCLUSION

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.

摘要

目的

开发一种实用、简单且稳健的多位点饱和诱变（MSSM）方法，该方法能够同时对氨基酸位点进行重组，以生成聚焦突变文库。

结果

一种基于延长重叠延伸PCR（POE-PCR）和简单克隆技术的通用无限制酶和无连接酶的MSSM方法（简单MSSM）。作为简单MSSM原理的证明，增强型绿色荧光蛋白（eGFP）基因被用作模板基因，用于同时诱变五个密码子。对48个随机选择的克隆进行测序。测序结果显示，所有48个克隆均至少显示一个突变密码子（突变效率 = 100%），48个克隆中有46个在所有五个密码子处均发生突变。在这五个密码子处获得的多样性分别为27、24、26、26和22，分别对应于NNK简并（32个密码子；NNK，K = T或G）提供的理论多样性的84%、75%、81%、81%、69%。