(515v) Towards Production Of Recombinant Silica-Precipitating Peptides By Understanding Biosilicification In Diatoms

Diatoms, unicellular photosynthetic algae, have ornate species-specific cell walls made of amorphous silica. Several classes of diatoms exhibit fractal pores ranging in size from 10 to 1000 nm in diameter. There is great curiosity in the scientific field about the processes involved in the creation of these intricate patterns because they are far superior to man-made materials. Unfortunately there is limited knowledge about the mechanism of biosilicification. Modified peptides called silaffins are one of the agents responsible for silica deposition in the marine diatom Cylindrotheca fusiformis. Silaffins are produced by the proteolytic processing of the polypeptide sil1p (encoded by the sil1 gene). Polyamine chains are attached to the lysine residues of silaffins and the serine residues are phosphorylated. The enzymes responsible for these post-translational modifications are unknown. The overall aim of this work is to determine how silaffins are created in C. fusiformis and to use this information to produce recombinant silaffins. These silaffins can subsequently be used to make novel biomaterials at ambient temperature and pressure for applications such as enzyme immobilization, biosensing, high surface area catalysis, etc. The work presented here is the purification of the protease involved in generating silaffins and the production of recombinant sil1p. Escherichia coli was used as the host for expressing recombinant sil1p. Sil1p was purified using immobilized metal ion affinity chromatography and its identity was confirmed using mass spectrometry. The protease responsible for cleaving sil1p was purified using standard biochemical fractionation techniques. The assay used for detecting protease activity involves the use of intramolecularly quenched fluorogenic substrates. The recognition motif separates a fluorescent donor group from a fluorescence quencher group. Protease activity was monitored by measuring the increase in fluorescence and the cleavage site was verified by identifying the peptide fragments using Fourier transform ion cyclotron resonance mass spectrometry. Isolation of the protease responsible for cleaving sil1p was achieved by using a combination of ion exchange chromatography, affinity chromatography and gel filtration. The protease was used to generate silaffins from recombinant sil1p.