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Directed self-immobilization of alkaline phosphatase on micro-patterned substrates via genetically fused metal-binding peptide.

Authors
  • Kacar, Turgay1
  • Zin, Melvin T
  • So, Christopher
  • Wilson, Brandon
  • Ma, Hong
  • Gul-Karaguler, Nevin
  • Jen, Alex K-Y
  • Sarikaya, Mehmet
  • Tamerler, Candan
  • 1 Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA.
Type
Published Article
Journal
Biotechnology and Bioengineering
Publisher
Wiley (John Wiley & Sons)
Publication Date
Jul 01, 2009
Volume
103
Issue
4
Pages
696–705
Identifiers
DOI: 10.1002/bit.22282
PMID: 19309754
Source
Medline
Language
English
License
Unknown

Abstract

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self-assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site-specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N-terminus of AP (nGBP1-AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi-functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple-repeat constructs, 5GBP1-AP displayed the best bi-functional activity and, therefore, was chosen for self-immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1-AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self-immobilization of the bi-functional enzyme on micro-patterned substrates where genetically linked 5GBP1-AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site-specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic-binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. (c) 2009 Wiley Periodicals, Inc.

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