Directed Self-Immobilization of Alkaline Phosphatase on Micro-Patterned Substrates Via Genetically Fused Metal-Binding Peptide


Kacar T., ZIN M. T. , SO C., WILSON B., MA H., Gul-Karaguler N. , ...More

BIOTECHNOLOGY AND BIOENGINEERING, vol.103, no.4, pp.696-705, 2009 (Journal Indexed in SCI) identifier identifier

  • Publication Type: Article / Article
  • Volume: 103 Issue: 4
  • Publication Date: 2009
  • Doi Number: 10.1002/bit.22282
  • Title of Journal : BIOTECHNOLOGY AND BIOENGINEERING
  • Page Numbers: pp.696-705

Abstract

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self-assembly and case 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 (GBPI) 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. Biotechnol. Bioeng. 2009;103: 696-705. (c) 2009 Wiley Periodicals, Inc.