Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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In Vitro Selection of High Affinity DNA-Binding Protein Based on Plasmid Display Technology

  • Choi, Yoo-Seong (School of Chemical and Biological Engineering, Seoul National University) ;
  • Joo, Hyun (Department of Molecular Physiology, College of Medicine, Inje University) ;
  • Yoo, Young-Je (School of Chemical and Biological Engineering, Seoul National University)
  • Published : 2005.10.01
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Abstract

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

Keywords

References

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