A new approach for purification of the catalytic site of the angiotensin-conversion enzyme, N-domain, mediated by the ELP-Intein system.

Angiotensin-converting enzyme I (ACE) is a key part of the renin-angiotensin system. Its main function is to regulate blood pressure and the balance of salts in the body. Somatic ACE has two domains, N-C-, each of which has a catalytic site that exhibits 60%sequence identity. The N-domain has a specific action in the hydrolysis of beta-amyloid bodies and angiotensin (1-7), which activates the MAS receptor and triggers anti-thrombotic and anti-inflammatory actions. Our goal was to obtain the catalytic site Ala361 to Gly468 of the N domain region, csACEN, without needing purification by chromatography. We employed a method that uses an Elastin-like Polypeptide (ELP) and Intein sequences linked to the peptide of interest. The more differential for obtaining the pure peptide was the cultivation temperatures in the synthesis of ELPcsACEN at 37 °C, with a significant increase in expression. In the purification by ELP precipitation, we recorded the highest efficiency in the concentrations of 0.57 M and 0.8 M of ammonium sulfate buffer. Intein autocleavage study allows removal of the ELP sequence at acidic pH, with the buffers MES and Tris-HCl The present study defined the best conditions for obtaining pure csACEN that the literature has not yet described for peptides. Obtaining pure csACEN aims at future studies for therapeutic use in hypertension, Alzheimer's, and oncology.

PRP8 Intein in Onygenales: Distribution and Phylogenetic Aspects.

Inteins (internal proteins) are mobile genetic elements, inserted in housekeeping proteins, with self-splicing properties. Some of these elements have been recently pointed out as modulators of genetic expression or protein function. Herein, we evaluated, in silico, the distribution and phylogenetic patterns of PRP8 intein among 93 fungal strains of the order Onygenales. PRP8 intein(s) are present in most of the species (45/49), mainly as full-length inteins (containing both the Splicing and the Homing Endonuclease domains), and must have transferred vertically in all lineages, since their phylogeny reflects the group phylogeny. While the distribution of PRP8 intein(s) varies among species of Onygenaceae family, being absent in Coccidioides spp. and present as full and mini-intein in other species, they are consistently observed as full-length inteins in all evaluated pathogenic species of the Arthrodermataceae and Ajellomycetaceae families. This conservative and massive PRP8 intein presence in Ajellomycetacean and Arthrodermatecean species reinforces the previous idea that such genetic elements do not decrease the fungal fitness significantly and even might play some role in the host-pathogen relationship, at least in these two fungal groups. We may better position the species Ophidiomyces ophiodiicola (with no intein) in the Onygenaceae family and Onygena corvina (with a full-length intein) as a basal member in the Arthrodermataceae family.

Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification.

Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis.

PRP8 intein in cryptic species of Histoplasma capsulatum: evolution and phylogeny.

The PRP8 intein is the most widespread intein among the Kingdom Fungi. This genetic element occurs within the prp8 gene, and is transcribed and translated simultaneously with the gene. After translation, the intein excises itself from the Prp8 protein by an autocatalytic splicing reaction, subsequently joining the N and C terminals of the host protein, which retains its functional conformation. Besides the splicing domain, some PRP8 inteins also have a homing endonuclease (HE) domain which, if functional, makes the intein a mobile element capable of becoming fixed in a population. This work aimed to study (1) The occurrence of this intein in Histoplasma capsulatum isolates (n=99) belonging to different cryptic species collected in diverse geographical locations, and (2) The functionality of the endonuclease domains of H. capsulatum PRP8 inteins and their phylogenetic relationship among the cryptic species. Our results suggest that the PRP8 intein is fixed in H. capsulatum populations and that an admixture or a probable ancestral polymorphism of the PRP8 intein sequences is responsible for the apparent paraphyletic pattern of the LAmA clade which, in the intein phylogeny, also encompasses sequences from LAmB isolates. The PRP8 intein sequences clearly separate the different cryptic species, and may serve as an additional molecular typing tool, as previously proposed for other fungi genus, such as Cryptococcus and Paracoccidioides.

Intein-mediated expression of cecropin in escherichia coli

Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.

PRP8 intein in Ajellomycetaceae family pathogens: sequence analysis, splicing evaluation and homing endonuclease activity.

Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications.

Inteins or 'internal proteins' are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride / Clonagem molecular do gene quitinase 33 (chit33) em Trichoderma atroviride

In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli.

Neste estudo Trichoderma atroviride foi escolhido como superprodutor da enzima quitinase dentre 30 isolados de Trichoderma sp. com base na atividade específica de quitinase. Clones de cDNA e genômico codificando chit33 foram obtidos deste isolado e seqüenciados. A comparação das seqüências genômica e de cDNA para definir a estrutura do gene indicou que este contém três pequenos introns e uma fase aberta de leitura codificando uma proteína de 321 aminoácidos. A seqüência de aminoácidos deduzida inclui um possível peptídio sinal de 19 aminoácidos. Homologia entre esta seqüência e outras proteínas Chit33 descritas de Trichoderma é discutida. A seqüência codificadora do gene chit33 foi clonada no vetor de expressão pET26b(+) e expressa em E. coli.

Phylogenetic analysis of PRP8 intein in Paracoccidioides brasiliensis species complex.

A recent species status investigation of the pathogenic fungus Paracoccidioides brasiliensis suggested the existence of three cryptic species. In the present study, the sequences of the PRP8 intein from P. brasiliensis isolates belonging to the three described genetic groups and two unidentified isolates were determined and analyzed in order to check their functionality and usefulness for species identification. All the isolates presented a full-length intein, although the Endonuclease domain seems to be inactive due to substitutions in the second essential aspartic acid residue. Phylogenetic analysis by Maximum-Parsimony, Maximum Likelihood, and Bayesian analysis clearly separated the isolates from the three species and revealed a significant difference between the Pb01 isolate and the remaining ones. The Pb01 isolate does not belong to any of the groups previously described since it presented a high divergence level compared to the three different genetic groups, corroborating some previous studies that suggested this isolate represents a new species of Paracoccidioides.

Sequence and annotation of the 369-kb NY-2A and the 345-kb AR158 viruses that infect Chlorella NC64A.

Viruses NY-2A and AR158, members of the family Phycodnaviridae, genus Chlorovirus, infect the fresh water, unicellular, eukaryotic, chlorella-like green alga, Chlorella NC64A. The 368,683-bp genome of NY-2A and the 344,690-bp genome of AR158 are the two largest chlorella virus genomes sequenced to date; NY-2A contains 404 putative protein-encoding and 7 tRNA-encoding genes and AR158 contains 360 putative protein-encoding and 6 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands, and intergenic space is minimal. Two of the NY-2A genes encode inteins, the large subunit of ribonucleotide reductase and a superfamily II helicase. These are the first inteins to be detected in the chlorella viruses. Approximately 40% of the viral gene products resemble entries in the public databases, including some that are unexpected for a virus. These include GDP-d-mannose dehydratase, fucose synthase, aspartate transcarbamylase, Ca(++) transporting ATPase and ubiquitin. Comparison of NY-2A and AR158 protein-encoding genes with the prototype chlorella virus PBCV-1 indicates that 85% of the genes are present in all three viruses.