Expression and characterization of a new valosin-containing protein from silkworm

Valosin-containing protein (VCP) is a type-II adenosine triphosphatase (ATPase) wih extensive biological function in organisms. Silkworm is the second insect model for genetic studies and a bioreactor for proteinaceous drugs and biomaterials. In this paper, a new VCP-like gene was amplified from the fat body of silkworm following genome prediction and spliced expressed sequence tag sequences, using both reverse transcription polymerase chain reaction (RT-PCR) and 3'-RACE (rapid amplification of complementary DNA ends) methods. Bioinformatical analysis showed that the translated amino acid sequence contained a highly conserved domain of VCPs similar to that of many insects. This domain consists of the conserved structure motifs of the ATP binding site and the catalytical center, which is closely related to the insect VCPs in a phylogenetic tree. The silkworm VCP-like gene was successfully inserted into the plasmid and transformed into Escherichia coli cells to express VCP-like protein with ATPase activity. The expression of silkworm VCP-like protein was also confirmed by Western blotting and mass spectrometric analyses. Distribution of the VCP-like gene in various tissues of the silkworm was also studied by real-time PCR. Results showed that the messenger RNA (mRNA) of VCP-like protein is widely expressed in fat body, reproductive organs (testis or ovary), silk gland, head, Malpighian tubule, epidermis and midgut. Among them, fat body has the highest mRNA expression level of the VCP-like gene, while the midgut has the lowest expression level. This study provides groundwork for further study on the structure and function of the new VCP-like protein.