title: Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.
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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Feng
authors.initials: F
authors.lastname: Cheng
authors.affiliation: 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.
authors.forename: Jian-Miao
authors.initials: JM
authors.lastname: Xu
authors.affiliation: 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.
authors.forename: Chao
authors.initials: C
authors.lastname: Xiang
authors.affiliation: 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.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.affiliation: College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, 518060, People's Republic of China.
authors.forename: Li-Qing
authors.initials: LQ
authors.lastname: Zhao
authors.affiliation: 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. zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D014170:Transformation, Genetic
chemical_list: D000596:Amino Acids; D003062:Codon; D009841:Oligonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Multi-site saturation mutagenesis; POE-PCR; Protein engineering; Simple-MSSM