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Expression and functional analysis of Fas ligand‐like protein from silkworm, Bombyx mori
Author(s) -
Yanagida Kosuke,
Yabu Takeshi,
Shiba Hajime
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.647.8
Subject(s) - bombyx mori , microbiology and biotechnology , biology , affinity chromatography , western blot , recombinant dna , pichia pastoris , size exclusion chromatography , complementary dna , apoptosis , protein subunit , biochemistry , enzyme , gene
In the silkworm, Bombyx mori , the degradation of larval tissues is an essential process for generating raw materials to build an adult body. It has been shown that most of the larval tissues disappear in the pupal stage upon activation of Bmcaspase dependent apoptosis. As an approach to identify the ligand responsible for initiating the death pathway, we searched Silk Base database and cloned a candidate cDNA fragment encoding a protein sequence similar to the mammalian Fas ligand in the Pichia Pastoris yeast expression system. The expressed recombinant protein was purified using Ni‐agarose affinity column and Sephacryl S‐300 gel filtration column. While the gel filtration column chromatography gave a molecular weight of 108,000, the subunit molecular weight was 38,000 as estimated by using SDS‐PAGE under reducing condition, indicating that the protein was purified as a homo‐trimer. To verify its capability of inducing apoptosis, the recombinant protein was injected into the larvae of V6 (6th day after entering fifth instar) at various amounts. All larvae died within 24 hours at the dosage of 16 ng or more protein. Inside the dead bodies, the lysis of the larval tissues was observed and a remarkable elevation of Bmcaspase activity was detected; Ac‐DEVD‐MCA, an artificial fluorescent substrate, was used. Western blot analysis revealed the occurrence of BmJNK phosphorylation in the same tissues. Our results clearly showed that the purified recombinant protein is able to induce Bmcaspase‐dependent apoptosis that causes disappearance of larval tissues, possibly through BmJNK signaling pathway.