Bioengineering of the silica-polymerizing enzyme silicatein-alpha for a targeted application to hydroxyapatite.
Since its discovery, numerous biotechnological approaches have aimed to explore the silica-polymerizing catalytic activity of the enzyme silicatein. In vivo, silicatein catalyzes polymerization of amorphous silica nanospheres from soluble precursors. In vitro, it directs the formation of nanostructured biosilica. This is of interest for various applications that strive to benefit from both the advantages of the biological system (i.e., silica synthesis under physiological conditions) and the cell mineralization-stimulating effect of biosilica. However, so far immobilization of silicatein has been hampered by the complex multistep procedure required. In addition, the chemical surface modifications involved not only restrict the choice of carrier materials but also render application of silicatein to hydroxyapatite (HA) of mineralized tissue impossible. Here we describe the bioengineering of silicatein, adapted for application in the fields of bone regeneration, tissue engineering, and dental care. Inspired by Glu-rich sequences of mammalian proteins that confer binding affinity to HA, a novel protein-tag was developed, the Glu-tag. Following expression of Glu-tagged silicatein, the HA-binding capacity of the enzyme is demonstrated in combination with synthetic and dental HA. Furthermore, immobilized Glu-tagged silicatein catalyzes synthesis of biosilica coatings on both synthetic HA nanofibrils and dental HA. Hence, Glu-tagged silicatein reveals a considerable biomedical potential with regenerative and prophylactic implementations.
Institute for Physiological Chemistry and Pathobiochemistry, Medical School, Johannes Gutenberg-University, Duesbergweg 6, 55128 Mainz, Germany.
Filipe
F
Natalio
Thorben
T
Link
Werner E G
WE
Müller
Heinz C
HC
Schröder
Fu-Zhai
FZ
Cui
Xiaohong
X
Wang
Matthias
M
Wiens
D000818:Animals; D057005:Bioengineering / Q000379:methods*; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D003460:Crystallization; D017886:Durapatite / Q000138:chemical synthesis*; D004800:Enzymes, Immobilized / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D057139:Nanofibers / Q000648:ultrastructure; D011108:Polymers / Q000138:chemical synthesis*; D011485:Protein Binding; D012822:Silicon Dioxide / Q000138:chemical synthesis*; D047233:Suberites / Q000201:enzymology; D034421:Sus scrofa
D004800:Enzymes, Immobilized; D011108:Polymers; C112571:silicatein alpha; D018698:Glutamic Acid; D012822:Silicon Dioxide; D017886:Durapatite; D002403:Cathepsins