Recombinant Expression, Purification And Chitin Binding Specificity Of Different CPAP3s From Bombyx Mori

Insect cuticle, a crucial structure that protects insects against chemical injuries, physical damage and pathogen infection, is mainly composed of chitin embedded in proteins with chitin binding activities. Previously, our proteomic analysis of molting fluid from Bombyx mori identified six unique proteins with three family 14 chitin binding modules called CPAP3 (cuticle protein analogue to peritrophin with 3 chitin binding domains), which were supposed to be released into molting fluid from the old cuticle and had been proved to be important for the structural integrity of the cuticle in Tribolium castaneum. Therefore, this work aiming chitin binding specificity of different BmCPAP3s will provide basic understanding on how BmCPAP3s will function during chitin formation on protein level.The work in this thesis includes:(1) The cDNA sequences of BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned by PCR. The lengths of open reading frames of BmCPAP3s were 714 bp,684 bp,873 bp,789 bp,645 bp and 621 bp, respectively.BmCPAP3s were predicted to be secreted proteins with their N-terminal carrying signal peptides. The development-specific expression pattern showed that BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B and BmCPAP3-C were mainly expressed at pupa, meanwhile, BmCPAP3-D1 and BmCPAP3-D2 were highly expressed at 24 hours before adult and pupa day-1, respectively. These investigations indicated that BmCPAP3s might play a vital role during pupation.(2) The expression vector of BmCPAP3s were constructed and introduced into E.coil BL21 cells, then induced by IPTG. The target proteins were detected from supernatant of the cell lysis and were purified by chelate affinity chromatography.(3) Their binding activities towards solid chitin, colloidal chitin and partly deacetylated chitin (chitosan) were investigated. The results demonstrated that all six ?CPAP3s could bind to solid chitin, colloidal chitin and chitosan. However, they showed very strong affinity (more than 80% bind) only towards solid chitin, weaker affinity towards colloidal chitin (40% bind) and very weak affinity towards chitosan (less than 30% bind), suggesting that they prefer to bind acetylated crystalline chitin.(4) The molecular weight of BmCPAP3-C was 53.4 kD in solution, and it was double of the predicted value. Five truncated proteins of BmCPAP3-C were constructed and expressed in E.coil. However, the recombinant proteins were existed in inclusion body and could not be purified.