Purification And Identification Of The Chitin-binding Proteins From The Larval Cuticlar Proteins Of Silkworm, Bombyx Mori

The silkworm, Bombyx mori, as a completely domesticated insect with good breeding and genetic system, has been considered as an ideal model for Lepidoptera. In 2004, the genomic project of B. mori was completed by Chinese and Japanese groups. The hypothetical protein database of silkworm was constructed with a large number of genomic and EST sequences, which provide us a database for proteomics searching.The insect cuticle is an extracellular structure covering the total outer surface of the animal and providing protection against the harm from the environment. It plays essential roles in multiple physiological functions to protect its body from the invasion of pathogens, the penetration of insecticides, physical injury, and dehydration. The varying mechanical properties of cuticles provide the optimal environment during all developmental stages of insect.Insect cuticle is mainly composed of chitin and proteins, with small amounts of lipid. Cuticular protein and chitin self-assemble into regularly arranged fibrils, which is the fundamental structure of cuticle. Several sequence motifs have been identified even in cuticular proteins from distantly related species and such conserved motifs play important roles for the physical properties of cuticle. The most important motif is the "R&R Consensus " identified by Rebers and Riddiford. Evidence showed that proteins with "R&R" motif bound to chitin, but recombinant CPF proteins did not bind to chitin.In this study, SDS was used to extracted cuticular proteins from the silkworm larvae. After dialysis to remove SDS, the dialysate was used for binding assay. Further LC-MS/MS analysis was carried out and 90 unique peptides were identified. A total of 85 peptides matched to 22 proteins, including nine cuticular proteins, two lysozyme precursors, two proteins with chitin-binding-type 2 domain, two ATP synthases, a 30 K protein, an apolipophorinâ…¢, a lipase, a promoting protein, a chitinase-like protein, a muscular protein 20, and an unknown protein. 38 peptides matched to nine cuticular proteins, with an average 23% sequence coverage. These cuticular proteins included seven RR-1 proteins, a RR-3 protein, and a Tweedle protein.BmCPR56 and BmorCPTl, which have RR-1 and Tweedle domain, respectively, were chosen to be expressed in E. coli. The two recombinant proteins were purified and subjected to in vitro chitin-binding assay. The cDNA sequences of the two genes were cloned with specific primers, inserted into the expression vector PGEX-4T-1 and the vector constructions were confirmed by sequencing. E. coli BL21 (DE3) was used as an expression host strain and the target proteins were induced with IPTG. The target proteins were released from the cells by sonication and further purified with GST column. The expressed Bmcpr56 has 166 amimo acids and the molecular weight of BmorCPR56-GST is about 45KD. And recombinant BmCPT1 has 460 amino acids and the molecular weight of BmorCPT1-GST is about 77KD. Rebers and Willis reported that GST-tag did not bind to the chitin, so the fusion proteins were directly used for binding assays. The results showed that the recombinant BmorCPR56 and BmorCPT1 bound to chitin in vitro for the first time.