Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.     

Plasmodium falciparum protein phosphatase type 1 functionally complements a glc7 mutant in Saccharomyces cerevisiae

We have identified a new homologue of protein phosphatase type 1 from Plasmodium falciparum, designated PfPP1, which shows 83-87% sequence identity with yeast and mammalian PP1s at the amino acid level. The PfPP1 sequence is strikingly different from all other P. falciparum Ser/Thr phosphatases cloned so far. The deduced 304 amino acid sequence revealed the signature sequence of Ser/Thr phosphatase LRGNHE, and two putative protein kinase C and five putative casein kinase II phosphorylation sites. Calyculin A, a potent inhibitor of Ser/Thr phosphatase 1 and 2A showed hyperphosphorylation of a 51kDa protein among other parasite proteins. Okadaic acid on the other hand, was without any effect suggesting that PP1 activity might predominate over PP2A activity in intra-erythrocytic P. falciparum. Complementation studies showed that PfPP1 could rescue low glycogen phenotype of Saccharomyces cerevisiae glc7 (PP1) mutant, strongly suggesting functional interaction of PfPP1 and yeast proteins involved in glycogen metabolism.     

Clonorchis sinensis: expression, characterization, immunolocalization and serological reactivity of one excretory/secretory antigen-LPAP homologue

From a Clonorchis sinensis adult cDNA plasmid library, a cDNA clone encoding a novel lysophosphatidic acid phosphatase (LPAP) homologue was isolated. The predicted molecular weight of putative protein was 48.8 kDa and the deduced amino acid sequence had 45%, 32%, and 29% identity with LPAP of Schistosoma japonicum, Danio rerio, and Homo sapiens, respectively. Prediction of signal peptide and Western blot analysis indicated that the CsLPAP homologue was an excretory-secretory antigen (ES antigen) of C. sinensis. Immunostaining revealed that the CsLPAP was markedly localized in the intestinal cecum, seminal receptacle and eggs of the adult worm. The recombinant CsLPAP showed slightly higher sensitivity (82.14%) and specificity (85.86%) than the crude worm antigen by enzyme-linked immunosorbent assay (ELISA), a result which suggested that the recombinant antigen might be valuable in the serodiagnosis of human clonorchiasis.     

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ∼60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).     

Isolation and Characterization of the PKAr Gene From a Plant Pathogen,Curvularia lunata

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.     

cDNA,genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda

αB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth, Antheraea pernyi.

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

The 110kDa glutathione transferase of Yarrowia lipolytica is encoded by a homologue of the TEF3 gene from Saccharomyces cerevisiae: cloning, expression, and homology modeling of the recombinant protein

The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

Gnathostoma spinigerum: Molecular cloning, expression and characterization of the cyclophilin protein

In this study, a cDNA encoding cyclophilin (CyP) of Gnathostoma spinigerum was cloned into a prokaryotic expression vector and expressed in Escherichia coli. The predicted molecular mass of the putative protein was 18.6 kDa, and the deduced amino acid sequence had 86, 84.8, 81.3 and 77.2% identity with the CyP of Dirofilaria immitis, Brugia malayi, Onchocerca volvulus and Caenorhabditis elegans, respectively. A prediction of linear B-cell epitopes with high hydrophilicity and immunoblotting results indicated that the recombinant CyP has antigenicity to humans. The recombinant CyP protein reacted with human gnathostomiasis sera but not with other parasitosis or healthy control sera, suggesting that it might be useful for the serodiagnosis of human gnathostomiasis.     

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis

A sodium-dependent phosphate transporter gene, DvSPT1, was isolated from a cDNA library using a probe derived from a subtracted cDNA library of Dunaliella viridis. Sequencing analyses revealed a cDNA sequence of 2649 bp long and encoded an open-reading frame consisting of 672 amino acids. The deduced amino acid sequence of DvSPT1 exhibited 31.2% identity to that of TcPHO from Tetraselmis chui. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa. Northern blot analysis indicated that the DvSPT1 expression was induced upon NaCl hyperosmotic stress or phosphate depletion. Functional characterization in yeast Na+ export pump mutant G19 suggested that DvSPT1 encoded a Na+ transporter protein. The gene sequence of GDvSPT1 (7922 bp) was isolated from a genomic library of D. viridis. Southern blot analysis indicated that there exist at least two homologous genes in D. viridis.     

cDNA cloning, expression, and characterization of an arylphorin-like hexameric storage protein, AgeHex2, from the mulberry longicorn beetle, Apriona germari

An arylphorin-like hexameric storage protein, AgeHex2, cDNA was cloned from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae), larval cDNA library. The complete cDNA sequence of AgeHex2 is comprised of 2,088 bp encoding 696 amino acid residues. The AgeHex2 had four potential N-glycosylation sites. The AgeHex2 contained the highly conserved two larval storage protein signature motifs. The deduced protein sequence of AgeHex2 showed high homology with A. germari hexamerin1 (51% amino acid identity), Tenebrio molitor hexamerin2 (49% amino acid identity), T. molitor early-staged encapsulation inducing protein (43% amino acid identity), and Leptinotarsa decemlineata diapause protein1 (43% amino acid identity). Phylogenetic analysis further confirmed the AgeHex2 is more closely related to coleopteran hexamerins than to the other insect storage proteins. Northern blot analysis confirmed that the AgeHex2 showed fat body-specific expression. The cDNA encoding AgeHex2 was expressed as a 75-kDa protein in the baculovirus-infected insect cells. Furthermore, N-glycosylation of the recombinant AgeHex2 was revealed by tunicamycin to the recombinant virus-infected Sf9 cells, demonstrating that the AgeHex2 is N-glycosylated. Western blot analysis using the polyclonal antiserum against recombinant AgeHex2 indicated that the AgeHex2 corresponds to a 75-kDa storage protein present in the A. germari larval hemolymph.     

Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen.

The second major allergen of Chamaecyparis obtusa (Japanese cypress) pollen, Cha o 2, has been purified and its cDNA cloned. Of patients with pollinosis caused by C. obtusa, 82.5% produce IgE antibodies which react with purified Cha o 2. The purified protein has a molecular mass of 46 kDa and its 12 N-terminal amino acid sequence displays a high homology with that of Cry j 2, the second major allergen of Cryptomeria japonica pollen. cDNA clones coding for Cha o 2 have been isolated using Cry j 2 cDNA as a probe. Cha o 2 cDNA clones were sequenced and found to code a putative 50-residue signal sequence and a 464-residue mature protein with a molecular weight of 50 kDa. Two possible N-linked glycosylation sites were found in the sequence. The deduced amino acid sequence of Cha o 2 shows 74.3% identity with that of Cry j 2. In its primary structure, Cha o 2 shows significant identity with those of the polygalacturonases of avocado, tomato, and maize as well as Cry j 2.