Abstract
Leucine-rich repeat (LRR) proteins play important roles in the transduction of cellular signals and activation of defense responses. By scanning the cDNA library of silkworm (Bombyx mori) pupae constructed in our laboratory, we identified a 1557 bp gene that encodes a protein homologous to the death-associated small cytoplasmic leucine-rich protein, which was named as BmDSCLP. The full-length gene (GenBank accession no. FJ602779) contained a 642 bp open reading frame (ORF) encoding 213 amino acid residues. The ORF of this gene was inserted into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression plasmid and the fusion protein was expressed in Escherichia coli BL21(DE3) cells. The fusion protein was purified by Ni-affinity chromatography and fast protein liquid chromatography (FPLC) and its size was then, determined by liquid chromatography-mass spectrometry (LC/MS/MS) and found to be 27.74 kD. Polyclonal antibodies were raised by subcutaneous injection of the recombinant protein into New Zealand white rabbits and the titer reached 1:12800. Analysis of the subcellular localization of the BmDSCLP protein revealed that, the protein was localized in both the cytoplasm and nucleus, but the amount in the former was slightly higher than that in the latter. In addition, real-time fluorescence quantification polymerase chain reaction studies were conducted to investigate BmDSCLP transcription at different developmental stages and in different tissues of the fifth instar larva. The results indicated that, BmDSCLP is widely transcribed in different stages and tissues of the silkworm. Analysis of stage-specific transcription patterns indicated that, the transcriptional level of BmDSCLP was highest in adults and lowest in eggs. Analysis of tissue-specific transcription patterns revealed that, the transcriptional level of BmDSCLP was highest in genital organs and lowest in silk glands. These results suggest that BmDSCLP plays important roles in the reproductive development of B. mori. Key words: Bombyx mori, death-associated small cytoplasmic leucine-rich protein, prokaryotic expression, fluorescence quantification polymerase chain reaction.
Highlights
Leucine-rich repeats (LRRs) were originally discovered by Patthy (1987) and are found in proteins from a diverse array of organisms ranging from bacteria and plants to yeast and humans (Buchanan and Gay, 1996)
By scanning the cDNA library of silkworm (Bombyx mori) pupae constructed in our laboratory, we identified a 1557 bp gene that encodes a protein homologous to the death-associated small cytoplasmic leucine-rich protein, which was named as BmDSCLP
We cloned the BmDSCLP gene from silkworm pupa using bioinformatics and RT-polymerase chain reaction (PCR) methods and expressed the target protein in E. coli
Summary
Leucine-rich repeats (LRRs) were originally discovered by Patthy (1987) and are found in proteins from a diverse array of organisms ranging from bacteria and plants to yeast and humans (Buchanan and Gay, 1996). The three-dimensional (3D) structure of the LRR present in a complex of porcine ribonuclease inhibitor with ribonuclease has been determined (Kobe and Deisenhofer, 1993; Kobe and Deisenhofer, 1996). The crystal structure shows that, each LRR is composed of a β-sheet and an α-helix. The hairpin-like structure of the LRR is mainly formed by the β-sheet (LxxLxLxxN/CxL) and α-helix (xaxx ± a ± ± ± ± a A ± ± x ± ±) that are connected by a loop ring (Kobe and Kajava, 2001). It is possible that other LRR proteins may have a similar structure in their repeat region
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