Figure 6
Inhibition of calcium carbonate crystallization in vitro by incubation with recombinant Sv-Clf1. (A) Linear schematics of the vector design for producing the recombinant Sv-Clf1 in Pichia pastoris. The sequence encoding the ORF of Sv-Clf1 was inserted upstream the alpha mating factor, between EcoRI and NotI restriction sites on the pPIC9K vector, with a 6XHis tag at the N’-terminus. (B) Following geneticin resistance selection, three colonies were isolated and grown in small scale. The extracted protein was tested in a western blot analysis using an Anti-His antibody, showing all three colonies (1–3; L = molecular weight ladder) produced the expected size recombinant protein, with the highest yield in colony 1 which was produced in 1 L for the Calcium Carbonate precipitation assay. (C) Calcium Carbonate precipitation in the absence of protein and in the presence of a recombinant human insulin gives the same result of tightly packed calcite (top left inset). In the presence of the recombinant Sv-Clf1 the Calcium Carbonate precipitates in an amorphous shape, with larger crystals which occasionally form spherules (bottom right inset).
