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

Biotechnol Bioeng. 2009 Jul 1;103(4):696-705. doi: 10.1002/bit.22282.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism*
  • Binding Sites
  • Enzymes, Immobilized*
  • Gold / metabolism*
  • Immobilized Proteins / genetics
  • Immobilized Proteins / metabolism*
  • Microscopy, Atomic Force
  • Protein Binding
  • Recombinant Fusion Proteins / metabolism
  • Surface Plasmon Resonance


  • Enzymes, Immobilized
  • Immobilized Proteins
  • Recombinant Fusion Proteins
  • Gold
  • Alkaline Phosphatase