Chitin-binding Domain Research Articles

Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins

Sortases are enzymes mostly found in Gram-positive bacteria which cleave proteins site-specifically. This feature makes them a promising tool in molecular biology and biotechnology. In this study, using bacterial surface display of recombinant proteins and ability of sortase A in site-specifically cleavage of the amino acid sequences, a novel method for one-step purification of recombinant proteins was developed. Using computational program tools, a chimeric protein containing a metallothionein (mt) and chitin binding domain (ChBD) was attached to the C-terminal domain of the truncated outer membrane protein A (Lpp′-ompA) using sortase recognition site (amino acid residues: LPQTG) as a separator. The structure of the chimeric protein was simulated using molecular dynamics to determine if the LPQTG motif is accessible to the sortase active site. The designed chimeric protein was expressed and purified. The purified chimeric protein was also displayed on the surface of E. coli cells. Both purified chimeric protein and the E. coli cells displaying Lpp′-ompA-mt-ChBD carrier protein were then treated with sortase to evaluate the efficiency of sortase-mediated cleavage of purified chimeric protein as well as surface displayed-chimeric protein. It is shown that mt-ChBD protein was successfully cleaved and dissociated from Lpp′-ompA carrier and released into the medium after treatment with sortase in both recombinant protein and surface displayed-chimeric protein. The experimental results confirmed the molecular dynamics analysis results. The presented method could be regarded as a novel strategy for one step expression and purification of recombinant proteins.

Dual function of a secreted fungalysin metalloprotease in Ustilago maydis.

Fungalysins from several phytopathogenic fungi have been shown to be involved in cleavage of plant chitinases. While fungal chitinases are responsible for cell wall remodeling during growth and morphogenesis, plant chitinases are important components of immunity. This study describes a dual function of the Ustilago maydis fungalysin UmFly1 in modulation of both plant and fungal chitinases. Genetic, biochemical and microscopic experiments were performed to elucidate the invitro and in planta functions of U.maydis UmFly1. U.maydis ∆umfly1 mutants show significantly reduced virulence, which coincides with reduced cleavage of the maize chitinase ZmChiA within its chitin-binding domain. Moreover, deletion of umfly1 affected the cell separation of haploid U.maydis sporidia. This phenotype is associated with posttranslational activation of the endogenous chitinase UmCts1. Genetic complementation of the ∆umfly1 mutant with a homologous gene from closely related, but nonpathogenic, yeast fully rescued the cell separation defect invitro, but it could not recover the ∆umfly1 defect in virulence and cleavage of the maize chitinase. We report on the dual function of the secreted fungalysin UmFly1. We hypothesize that co-evolution of U.maydis with its host plant extended the endogenous function of UmFly1 towards the modulation of plant chitinase activity to promote infection.

An ungrouped cuticular protein is essential for normal endocuticle formation in the brown planthopper

Using transcriptome analysis of tissues of the brown planthopper (BPH), Nilaparvata lugens, we identified a gene tentatively designated NlCP21.92 that was expressed at high levels in the integument. Spatiotemporal expression profiling with quantitative PCR and Western blotting verified its integument-specific expression and showed periodic expression during molting. The open reading frame was GC-rich and encoded a hydrophobic polypeptide. The polypeptide contained AAPA/V repeat motifs and other sequence features similar to several reported cuticular proteins but lacked an R&R consensus and other chitin-binding domains. Double-stranded RNA–mediated RNA interference of the NlCP21.92 resulted in abnormal and lethal morphological phenotypes, and transmission electron microscopy revealed the corresponding ultrastructural defects. Immunohistochemical staining demonstrated that the NlCP21.92 protein was primarily located in the procuticle. Our results suggest that NlCP21.92 is a novel ungrouped cuticular protein essential for normal endocuticle formation.

Identification and characterization of chitin deacetylase2 from the American white moth, Hyphantria cunea (Drury)

Chitin deacetylases (CDAs) are enzymes that catalyze the conversion of chitin into chitosan, thereby influence the mechanical and permeability properties of structures such as the cuticle and peritrophic matrices. The full length cDNAs of chitin deacetylase2 (CDA2) genes from Hyphantria cunea were fully cloned by PCR amplification. Two cDNA sequences of HcCDA2 were searched from transcriptome of H. cunea and named as HcCDA2a and HcCDA2b. The deduced protein sequences showed that HaCDA2a and HaCDA2b are synthesized as preproteins of 524 and 518 amino acid residues with an 18-amino acid signal peptide, respectively. HcCDA2a and HcCDA2b contained a chitin-binding domain (ChBD), a low-density lipoprotein receptor class A domain (LDLa) and a polysaccharide deacetylase-like catalytic domain (CDA). Gene expression analyses results showed that HcCDA2a and HcCDA2b were both expressed at the head, integument, foregut, midgut, hindgut, Malpighian tubules and fat body, as well as the 1st to 5th days of fifth instar larvae. Western blot analyses revealed that HcCDA2 protein was highly abundant in the head and integument, and the developmental expression result in the fifth instars showed that HcCDA2 was highly present at the first two days. Besides, RT-PCR results showed that HcCDA2a and HcCDA2b were both expressed in integument and head, whether in molting stage or feeding stage. No visiable phenotypic changes were observed after injection of dsHcCDA2b, while lethal phenotypes of cuticle shedding failure and high mortality were resulted from injection of dsHcCDA2a. The silence of HcCDA2a leads to the ecdysis failure and death of H. cunea. These results suggest that HcCDA2 plays an important role during insect development, and provide new candidate targets and basis for developing environment-friendly pesticides.

Genome-wide identification of chitinase and chitin deacetylase gene families in the oriental fruit fly, Bactrocera dorsalis (Hendel)

Chitinases (Chts) and chitin deacetylases (CDAs) are important enzymes required for chitin metabolism in insects. In this study, 12 Cht-related genes (including seven Cht genes and five imaginal disc growth factor genes) and 6 CDA genes (encoding seven proteins) were identified in Bactrocera dorsalis using genome-wide searching and transcript profiling. Based on the conserved sequences and phylogenetic relationships, 12 Cht-related proteins were clustered into eight groups (group I–V and VII–IX). Further domain architecture analysis showed that all contained at least one chitinase catalytic domain, however, only four (BdCht5, BdCht7, BdCht8 and BdCht10) possessed chitin-binding domains. The subsequent phylogenetic analysis revealed that seven CDAs were clustered into five groups (group I–V), and all had one chitin deacetylase catalytic domain. However, only six exhibited chitin-binding domains. Finally, the development- and tissue-specific expression profiling showed that transcript levels of the 12 Cht-related genes and 6 CDA genes varied considerably among eggs, larvae, pupae and adults, as well as among different tissues of larvae and adults. Our findings illustrate the structural differences and expression patterns of Cht and CDA genes in B. dorsalis, and provide important information for the development of new pest control strategies based on these vital enzymes.

Cloning, characterization and substrate degradation mode of a novel chitinase from Streptomyces albolongus ATCC 27414

A novel chitinase gene was cloned from Streptomyces albolongus ATCC 27414, and expressed successfully in Escherichia coli BL21. The recombinant enzyme (SaChiA4) belongs to glycoside hydrolases (GH) family 18 and consists of a catalytic domain and a chitin binding domain (CBD) in its C-terminus. SaChiA4 was purified homogeneously (specific activity of 66.2 U/mg with colloidal chitin as substrate), and showed a molecular mass of approximately 47 kDa. SaChiA4 showed its optimal activity at pH 5.0 and 55 °C and exhibited remarkable pH and temperature stability. SaChiA4 has been proved to have a higher specificity toward glycosides containing acetyl groups and hydrolyzes the substrates in a non-processive manner with higher ability to produce (GlcNAc)2 and GlcNAc. The results indicated that SaChiA4 is a novel endo-type chitinase, which has potential applications in the treatment of chitin wastes and the production of (GlcNAc)2.

A chitinase from pacific white shrimp Litopenaeus vannamei involved in immune regulation

Chitinases are a group of hydrolytic enzymes that hydrolyze chitin and widely exist in organisms. Studies in mammals have demonstrated that chitinases play important roles in regulation of humoral and cellular immune responses. In arthropods, although it is well known that chitinases are involved in growth, molting and development, the current knowledge on the role of chitinases in immunity, especially in immune regulation, remains largely unknown. In this study, a chitinase (LvChi5) from Litopenaeus vannamei was representatively selected for studying its immune function. The start codon of LvChi5 was corrected by 5′RACE analysis and its protein sequence was reanalyzed. LvChi5 contains a catalytic domain and a chitin binding domain and shows no inhibitory effect on growth of bacteria in vitro. However, in vivo experiments demonstrated that silencing of LvChi5 increased the mortality of shrimp infected with white spot syndrome virus (WSSV) and Vibro parahaemolyticus and significantly upregulated the load of pathogens in tissues. The expression of various immune related genes, including transcription factors, antimicrobial peptides and other functional proteins with antibacterial and antiviral activities, was widely changed in LvChi5 silencing shrimp. Moreover, the recombinant LvChi5 protein could enhance the phagocytic activity of hemocytes against bacteria. These suggested that shrimp chitinase could play a role in regulation of both humoral and cellular immune responses in shrimp.

Molecular cloning and characterization of Pif gene from pearl mussel, Hyriopsis cumingii, and the gene expression analysis during pearl formation.

In the present study, the Pif gene of the freshwater pearl aquaculture mussel, Hyriopsis cumingii (HcPif) was successfully cloned and functionally characterized. The full sequence of HcPif gene consists of 3415 base pairs, which putatively encode two proteins, HcPif90 and HcPif80. A sequence analysis revealed that HcPif contained a von Willebrand factor type A domain and a chitin-binding domain, and shared many functional residues with other Pif homologues. A highly conserved sequence, FKGLDEIELML, at the C-terminus of Pif80s was identified as the key functional site. The corresponding peptide fragment markedly modified the morphology of calcite crystallites in CaCO3 crystallization assay and might play an essential role in the interactive binding between HcPif80 and CaCO3. Moreover, real-time PCR results showed that HcPif gene was dominantly expressed in the pearl secreting tissues and its expression changed in response to the different development status of the pearl sac during pearl aquaculture. The gene expression of HcPif was maximum 7days after mantle grafting and declined to about the control level on day 30. Our in vitro and in vivo experimental data indicated that HcPif gene possessed the inherent characteristics of a nacre formation gene and its expression might faithfully reflect the pearl secretion status of the pearl mussels examined. Our findings may extend the understanding of the biomineralization mechanism of nacre formation and provide a potential biomarker for pearl farming.

Molecular characterization and tissue expression analysis of five genes for chitinase in the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

Insect chitinases are hydrolytic enzymes that cleave chitin of the cuticle and peritrophic membrane during molting. Multiple genes encode insect chitinases, which are characterized as having diverse chemical and enzymatic properties depending on the time and the site of expression. This work was done to isolate and characterize chitinase genes from the red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver), a cryptic pest of many palm trees. The isolated five genes were phylogenetically clustered into five different groups (I, II, III, VI, and VII) of the glycoside hydrolase family 18 (GH18). Domain structure analysis revealed that RfCht1 (group I), RfCht3 (group VI), and RfCht5 (group VII) each retained a single catalytic domain of the GH18, whereas RfCht2 (group III)) and RfCht4 (group II) possessed two and five GH18 catalytic domains, respectively. RfCht1, RfCht2, and RfCht3 each retained a single chitin-binding domain (CBD) and RfCht4 retained five CBDs, but RfCht5 lacked CBD. Developmental and tissue expression profiles showed high levels of transcripts of the five genes in the newly hatched first instar larvae. RfCht1 and RfCht2 transcripts were expressed constitutively almost with high levels in young and mature eggs, in all tested larval instars, pre-pupae, pharate pupae, and adults; whereas RfCht3 and RfCht5 transcripts were expressed as low levels in the early instars larvae few hours before molting. In late developmental stages and mature eggs, RfCht3, RfCht4, and RfCht5 were expressed as low levels mainly in the cuticle. This study presents the first report on chitinase genes in the RPW and suggests that these genes have additional roles in the weevil development, which require further elucidation. Key words: Chitinase, conserved motifs, cuticle, domain structure, expression profile, Rhynchophorus ferrugineus

Crystal structures of an archaeal chitinase ChiD and its ligand complexes.

Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the C-terminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family.

CPAP3 proteins in the mineralized cuticle of a decapod crustacean

The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3’s, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3’s, suggested a major role for CPAP3’sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins –theoretically binding three chitin strands– was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.

Immune function against bacteria of chitin deacetylase 1 (EcCDA1) from Exopalaemon carinicauda

Chitin deacetylase (CDA, EC 3.5.1.41), belonging to a family of extracellular chitin-modifying enzymes, can catalyze the deacetylation of chitin. In this study, the full-length cDNA sequence encoding chitin deacetylase 1 (EcCDA1) was obtained fromExopalaemon carinicauda. The complete nucleotide sequence of EcCDA1 contained a 1611 bp open reading frame (ORF) encoding EcCDA1 precursor of 536 amino acids. The domain architecture of the deduced EcCDA1 protein contained a signal peptide, a chitin-binding peritrophin-A domain (ChtBD2), a low-density lipoprotein receptor class A domain (LDLa) and a Polysacc_deac_1 domain. EcCDA1 mRNA was predominantly expressed in the gills. The expression of EcCDA1 in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. The expression of EcCDA1 in the prawns challenged with V. parahaemolyticus was up-regulated at 12 h (p < 0.05), and significantly up-regulated at 24 h and 48 h (p < 0.01), and then returned to the control levels at 96 h post-challenge (p > 0.05). At the same time, the expression in Aeromonas-challenged group was significantly up-regulated at 12, 24 and 48 h (p < 0.01) and returned to the control levels at 120 h post-challenge (p > 0.05). Then, EcCDA1 was recombinantly expressed in Pichia pastoris and the purified recombinant EcCDA1 could not inhibit the growth of V. parahaemolyticus or A. hydrophila, which indicated that the CDA1 may play its biological activity in immune defense by deacetylation from chitin.

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm, Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome‐wide search for genes encoding proteins with peritrophin A‐type (ChtBD2) chitin‐binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein‐coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP‐encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co‐localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up‐regulated by 20‐hydroxyecdysone treatment. These results provide a genome‐level view of the chitin‐binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.

Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains

MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUNDThe insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation.OBJECTIVESTo determine the occurrence of alternative splicing in chitinases of L. longipalpis.METHODSWe sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA.FINDINGSWe showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae.MAIN CONCLUSIONSWe show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Structural analysis of group II chitinase (ChtII) catalysis completes the puzzle of chitin hydrolysis in insects

Chitin is a linear homopolymer of N-acetyl-β-d-glucosamines and a major structural component of insect cuticles. Chitin hydrolysis involves glycoside hydrolase family 18 (GH18) chitinases. In insects, chitin hydrolysis is essential for periodic shedding of the old cuticle ecdysis and proceeds via a pathway different from that in the well studied bacterial chitinolytic system. Group II chitinase (ChtII) is a widespread chitinolytic enzyme in insects and contains the greatest number of catalytic domains and chitin-binding domains among chitinases. In Lepidopterans, ChtII and two other chitinases, ChtI and Chi-h, are essential for chitin hydrolysis. Although ChtI and Chi-h have been well studied, the role of ChtII remains elusive. Here, we investigated the structure and enzymology of OfChtII, a ChtII derived from the insect pest Ostrinia furnacalis. We present the crystal structures of two catalytically active domains of OfChtII, OfChtII-C1 and OfChtII-C2, both in unliganded form and complexed with chitooligosaccharide substrates. We found that OfChtII-C1 and OfChtII-C2 both possess long, deep substrate-binding clefts with endochitinase activities. OfChtII exhibited structural characteristics within the substrate-binding cleft similar to those in OfChi-h and OfChtI. However, OfChtII lacked structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in OfChi-h. Nevertheless, the numerous domains in OfChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin-binding modules (OfChtII-B4C1) displayed activity toward insoluble polymeric substrates that was higher than those of OfChi-h and OfChtI. Our observations provide the last piece of the puzzle of chitin hydrolysis in insects.

Molecular Cloning and Phylogenetic Analysis of a Chitin Deacetylase Isolated from the Epidermis of the Red Snow Crab &amp;lt;i&amp;gt;Chionoecetes japonicas&amp;lt;/i&amp;gt;

Chitin deacetylase (CDA; EC 3. 5. 1. 41) catalyzes the deacetylation of chitin. In this study, we successfully cloned and sequenced a chitin deacetylase gene from the red snow crab Chionoecetes japonicas. By using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends, we obtained a 2141-bp amplicon containing a chitin deacetylase gene (CjCDA) from the epidermis of C. japonicas. The amplicon contains a 1575-bp open reading frame that is predicted to encode a 525-amino acid protein. The structure predicted from the deduced amino acid sequence included an N-terminal signal peptide, chitin-binding domain (CBD), low-density lipoprotein receptor class A domain (LDL-A), and catalytic domain. Comparative analysis of the deduced amino acid sequence of CjCDA revealed the highest homology (74%) to gastrolith protein 59 of Cherax quadricarinatus. We used RT-PCR to evaluate the expression of CjCDA in various tissues of C. japonicas, and we observed that CjCDA was expressed only in the epidermis. A phylogenetic analysis, using the amino acid sequences of CjCDA and other known chitin deacetylases, showed that CjCDA belonged to a group of crustacean chitin deacetylases. To our knowledge, this is the first study reporting the cDNA cloning of a chitin deacetylase from a crab.

Characterization of Chitinase from Bacillus thuringiensis Isolated from the Dynastid Beetle, Allomyrina dichotoma L.

Chitin is a linear polysaccharide that consists of β-1,4-linked N-acetyl-glucosamine (GlcNAc) and it forms the carbohydrate backbone of crustacean and insect exoskeletons. Chitinase has the ability to degrade these chitins to its product chitin oligosaccharides which are used in potential applications of biomedicine, nutrition and bio-technology. Bacillus thuringiensis is one of the well-known bacteria producing chitinase. In present study, we identified and purified the chitinase from B. thuringiensis PRCII. Chitinase activity assay using crap shell chitin and T. molitor chitin indicates that three chitinases approximately 37, 42, and 71 kDa were detected from B. thuringiensis PRCII. In addition, we cloned and expressed the 71 kDa chitinase from B. thuringiensis PRCII. These chitinases exhibited two chitinase protein activity 71 and 50 kDa in both native and rBtchitinase using crap shell and T. molitor chitin. On the basis of the in silco analysis of Btchitinase, Btchitinase lacks chitin binding domain. However, it exhibited the active chitinase activity, suggesting that chitin binding domain is not necessary for chitin hydrolysis.

Pilot production of the recombinant peptide toxin of Heteractis crispa as a potential analgesic by intein-mediated technology

APHC3 is an analgesic polypeptide that was found in the sea anemone (Heteractis crispa), and contains 56 amino acid residues. This polypeptide is of interest for the development of medications for diseases, associated with inflammatory or neuropathological processes, as well as its use as an analgesic. This work presents an innovative biotechnological method for APHC3 production. We have constructed a recombinant plasmid intended for biosynthesizing the fusion protein consisting of a chitin-binding domain, DnaB mini-intein from Synechocystis sp. capable of undergoing pH-dependent self-cleavage, and the target peptide. In the process of biosynthesis the fusion protein aggregates and forms the inclusion bodies that are welcomed since APHC3 is a cytotoxic peptide. The target peptide recovery process developed by us involves 3 chromatographic steps. The method developed by us enables to produce 940 mg of the recombinant APHC3 from 100 g of the inclusion bodies. The method is straightforward to implement and scale up. The recombinant APHC3 activity and effectiveness as an analgesic was proved by animal testing.

A cuticle protein from the Pacific white shrimp Litopenaeus vannamei involved in WSSV infection

White spot syndrome virus (WSSV) is a major viral pathogen in global shrimp farming, causing huge economic damage. Through penetrating the outer surface of the target tissues, WSSV enters into the cells of the target tissue to complete the replication process in the host. In the present study, a cuticle protein gene from Litopenaeus vannamei, designated as LvAMP13.4, was identified and proved to be involved in WSSV invasion. The deduced amino acid sequence of LvAMP13.4 contained a signal peptide and a conserved chitin-binding domain type 4 (ChBD4). This cuticle protein gene was mainly expressed in stomach, gill and epidermis. The expression level of LvAMP13.4 was significantly changed during WSSV infection. Silencing of LvAMP13.4 by dsRNA interference apparently reduced the mortality rate and the WSSV copy number in shrimp upon WSSV infection. Furthermore, yeast two-hybrid system and Co-IP assay were performed to confirm that LvAMP13.4 could interact with the major envelop protein VP24 of WSSV. These data indicated that LvAMP13.4 was involved in the invasion process of WSSV through interaction with VP24. The present results could provide new insights for us in understanding the role of host cuticle proteins during virus invasion.