Prokaryotic Expression And Localization Analysis Of Calcyphosphine From Silkworm(Bombyx Mori)

Bombyx mori calcyphosphine (BmCAPS) is a calcium binding protein containing two EF-hand conserved domains. BmCAPS was also predicted that it contains two types of phosphorylation sites by bioinformatics analysis. It is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. However, it is still unclear on the functions and mechanism of BmCAPS protein. By now, most researches of CAPS were focused on vertebrate rather than invertebrate. There is not any report in Bombyx mori.Calcyphosphine gene was screened from the cDNA library of silkworm pupae constructed by our labortary,and named BmCAPS. We analyzed the sequence by bioinformatic tools and found that it contains an open reading frame (ORF) of 720 bp encoding a protein of 239 amino acids with a predicted molecular mass of 26.874 kD and a theoretical pI of 4.68. The ORF of BmCAPS gene was amplified by PCR and inserted into the prokaryotic expression vector pET-28a(+). PCR and digestion with BamHâ… /Xhoâ… showed that the designed fragment was inserted correctly. The recombinant protein His-BmCAPS was expressed successfully in E.coli BL21 Star(DE3) after induced by IPTG. It was fused with His-tag and purified by affinity chromatography. The molecular weight of this fusion protein was 30.434 kD(His tag was weight of 3.56 kD, and BmCAPS was weight of 26.874 kD), characterized by mass-spectrum after being further purified on FPLC. This result proved that His-tag BmCAPS was expressed in the right form. In order to obtain polyclonal antibody, the purified fusion protein was used to immunize a male New Zealand rabbit. Then, the antiserum was harvested, the titer of the polyclonal antibody reaches over 1:6400 measured by indirect ELISA, and the recombinant protein was specifically recognized by antiserum in Western blotting analysis. We performed subcellular localization of BmCAPS protein in BmN cell and it was identified only in cytoplasm. The transcription level of mRNA and expression level of protein in different stages of Bombyx mori and different tissues of fifth instar larva was also analyzed by the real-time fluorescent quantificational PCR and Western blotting. We found that the transcription level of BmCAPS mRNA gradually increased from egg to moth in accord with the results of Western blotting. And the transcription level and protein expression level of spermary was highest compared to other tissues, but there was no message detected in the tissues of head, epidermis and silk glands by Western blotting. All the results above will provide us some evidences for further studies on the function of BmCAPS.