Biochemical and Biophysical Research Communications
Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph
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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