Cloning, expression and purification of the active from of endothelin converting enzyme-1(ECE1)

A enzima conversora de endotelina-1 (ECE 1) e uma metaloendoprotease de membrana que catalisa a conversao das formas inativas do peptideo big-endotelina para as formas ativas de endotelina. As endotelinas (EDN) sao potentes peptideos vasoconstntores formados por 21 aminoacidos e sao produzidos por celulas endoteliais vasculares. Os peptideos desta familia estao envolvidos na regulacao da pressao arterial e podem ter papel relevante em patologias cardiovasculares e renais. O passo final do bioprocessamento das endotelinas e a conversao do precursor inativo big-endotelina para o peptideo ativo, atraves da clivagem entre o Trp2l e Va1/I1e22 pela enzima conversora de endotelina (ECE). Neste estudo, o dominio catalitico da enzima conversora de endotelina-1 (ECE1) de cerebro de rato foi amplificado por rt-PCR e clonado em vetor plasmidial de transferencia pBSV-8His. A construcao pBSV-8His-ECE1 e o DNA do baculovirus BaculoGOLD foram transfectados em celulas de inseto Sf9 em cultura de celulas em suspensao. A proteina recombinante expressa foi purificada por cromatografia de afinidade em coluna de niquel. A proteina recombinante obtida foi submetida a desglicosilacao enzimatica, a western blotting contra o anticorpo antiHis tag e ao espectro de absorcao de dicroismo circular, comprovando-se a ocorrencia de modificacoes pos-traducionais da proteina, a obtencao da proteina recombinante estruturada e a identificacao da proteina clonada em fusao ao His-tag. O aumento de escala de expressao, acompanhado da otimizacao da homogeneidade da purificacao da proteina expressa, serao uteis para futuras caracterizacoes bioquimicas e estruturais de ECE 1BV UNIFESP: Teses e dissertaçõe

Shotgun sequencing of the human transcriptome with ORF expressed sequence tags

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription–PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome

A Transcript Finishing Initiative for Closing Gaps in the Human Transcriptome

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms