Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph

https://doi.org/10.1016/S0006-291X(03)01425-6Get rights and content

Abstract

In a previous study, we reported that silkworm hemolymph inhibits apoptosis and that the anti-apoptotic component in silkworm hemolymph is a 30K protein. In this study, the 30K protein encoded by 30Kc6 was expressed in Escherichia coli. The recombinant 30K protein was expressed as an inclusion body, and the inclusion body was separated and refolded by affinity column chromatography using a 6× His tag. We demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein. The recombinant 30K protein inhibited the virus- or chemical-induced apoptosis in human cells as well as insect cells. Apoptosis-inhibitory activity of recombinant 30K protein was comparable to that of whole silkworm hemolymph. The recombinant 30K protein can be effectively used to minimize cell death and consequently increase the productivity by extending the production time of host cells in commercial animal cell culture.

Section snippets

Materials and methods

Plasmid construction. A plasmid containing 30K protein cDNA (30Kc6, GenBank Accession No.: X54735) was kindly provided by S. Izumi (Department of Biology, Tokyo Metropolitan University). The 30K protein cDNA was amplified by PCR with a temperature profile of 95 °C for 1 min, 56 °C for 1 min, and 72 °C for 1.5 min. The forward and reverse primers were 5-AGA CAT ATG ACA CTT GCA CCA AGA ACT-3 and 5-CAA CTC GAG GTA GGG GAC GAT GTA CCA-3, respectively, which contain the NdeI and XhoI sites,

Results and discussion

A 30K protein (gene product of 30Kc6) originating from silkworm (B. mori) was expressed in E. coli. After cell harvesting, the cells were disrupted by sonication and the cell lysate was centrifuged. Supernatant and precipitate were analyzed by SDS–PAGE. The band of 30K protein with a molecular mass of approximately 30,000 Da is shown in the precipitate of lysate of the transformed E. coli strain BL21(DE3) (Fig. 1A). To confirm more precisely that the overexpressed protein was the 30K protein,

Acknowledgements

The authors acknowledge the financial support of the Korea Science and Engineering Foundation through the Nano Bio-Electronic and System Center, Seoul National University, Seoul, Korea.

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