Directed self-immobilization of alkaline phosphatase on micro-patterned substrates via genetically fused metal-binding peptide.
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.
Kacar
Turgay
T
Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA.
Zin
Melvin T
MT
So
Christopher
C
Wilson
Brandon
B
Ma
Hong
H
Gul-Karaguler
Nevin
N
Jen
Alex K-Y
AK
Sarikaya
Mehmet
M
Tamerler
Candan
C
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004800:Enzymes, Immobilized; D006046:Gold / Q000378:metabolism*; D055767:Immobilized Proteins / Q000235:genetics / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
D004800:Enzymes, Immobilized; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D006046:Gold; D000469:Alkaline Phosphatase