Use of a novel affinity tag selected with a bacterial random peptide library for improving activity retention of glutathione S-transferase adsorbed on a polystyrene surface

Takaharu Sakiyama, Shunsuke Ueno, Koreyoshi Imamura, Kazuhiro Nakanishi

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Aiming at developing a novel affinity tag for site-specific immobilization of functional proteins onto polystyrene (PS) surfaces, Escherichia coli random peptide display library was screened for dodecapeptides exhibiting a high affinity toward PS plates. The selected peptides were commonly rich in hydrophobic amino acid residues and had two or three basic amino acid residues. Adsorption and desorption experiments for one of the selected peptide named PS1 (KGLRGWREMISL) showed that it was well and irreversibly adsorbed onto PS latex particles. To study its performance as an affinity tag, PS1 was genetically fused to a model enzyme, glutathione S-transferase (GST), in several manners, and the fusion enzymes were compared to the original GST in terms of the adsorption behavior onto the PS latex particles as well as the specific activities before and after the adsorption. The fusion GSTs in solution showed lower specific activities than the original one, and their adsorption behaviors were also altered. In particular, the fusion of PS1 to the N-terminal region of GST resulted in severe losses both in the specific activity and in the adsorptive ability. However, two types of GSTs fused with PS1 at the C-terminal region were well adsorbed onto the PS latex particles, and their specific activities after the adsorption were significantly higher than the original GST adsorbed on the PS latex particles. The fusion of PS1 to the C-terminal region of GST was thus shown to reduce the activity loss upon the adsorption onto the PS latex particles.

Original languageEnglish
Pages (from-to)207-214
Number of pages8
JournalJournal of Molecular Catalysis B: Enzymatic
Volume28
Issue number4-6
DOIs
Publication statusPublished - Jun 1 2004

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Keywords

  • Affinity tag
  • Glutathione S-transferase
  • Polystyrene surface
  • Random peptide library
  • Site-specific adsorption

ASJC Scopus subject areas

  • Biochemistry
  • Catalysis
  • Process Chemistry and Technology

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