Abstract
Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.
Highlights
Silicateins are proteins that form biosilica enzymatically in siliceous sponges
Construction of the Fusion Gene—The segment of the S. domuncula silicatein-␣ cDNA, which encodes the precursor of the protein, the propeptide and the mature enzyme, was obtained by means of polymerase chain reaction (PCR) using the following primers; forward primer (Fwd): 5Ј-gtttcatatgGCAGCCCAGCCCAAGTTTG-3Ј and the reverse primer (Rev): 5Ј-cctactcgagTTATAGGGTGGGATAAGATGCATC-3Ј ([stop codon in bold] nt1024 to nt1001; XhoI site in the tail)
Silicatein-trigger Factor Fusion Protein—Silicatein was expressed in E. coli as a proenzyme fused to the trigger factor (Fig. 1) in the pCold TF DNA vector
Summary
Silicateins are proteins that form biosilica enzymatically in siliceous sponges. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG).
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