A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences
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Ai-Sheng Xiong
Abstract
Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.
©2007 by Walter de Gruyter Berlin New York
Articles in the same Issue
- FOXM1, a typical proliferation-associated transcription factor
- Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer
- A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences
- Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5)
- Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain
- SARS-CoV accessory protein 7a directly interacts with human LFA-1
- Overexpression and mass spectrometry analysis of mature human acid ceramidase
- Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes
- Macrophage paraoxonase 2 (PON2) expression is upregulated by unesterified cholesterol through activation of the phosphatidylinositol 3-kinase (PI3K) pathway
- Acknowledgment
- Contents Biological Chemistry, Volume 388, 2007
- Author Index
- Subject Index
Articles in the same Issue
- FOXM1, a typical proliferation-associated transcription factor
- Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer
- A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences
- Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5)
- Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain
- SARS-CoV accessory protein 7a directly interacts with human LFA-1
- Overexpression and mass spectrometry analysis of mature human acid ceramidase
- Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes
- Macrophage paraoxonase 2 (PON2) expression is upregulated by unesterified cholesterol through activation of the phosphatidylinositol 3-kinase (PI3K) pathway
- Acknowledgment
- Contents Biological Chemistry, Volume 388, 2007
- Author Index
- Subject Index
