Clonagem e express?o da sequ?ncia codificante para o pept?deo maduro LTx4 da aranha Lasiodora sp em sistema pET21b.

Pept?deos neurot?xicos encontrados em venenos animais t?m despertado um grande interesse de pesquisadores. A possibilidade de utiliz?-los como ferramentas moleculares para estudos em canais i?nicos, al?m do uso como bioinseticidas e/ou como biof?rmacos, tem impulsionado as pesquisas nessa ?rea. Experimentos com o veneno bruto da aranha caranguejeira Lasiodora sp mostraram que o mesmo atua em canais i?nicos de pequenos vertebrados e de invertebrados. Recentemente, constru?mos uma biblioteca de cDNA a partir dos mRNAs presentes na gl?ndula de veneno dessa aranha. A varredura desta biblioteca, usando a t?cnica de ELISA, e o sequenciamento de DNA, permitiram a identifica??o de cinco toxinas presentes no veneno, nomeadas LTx1, LTx2, LTx3, LTx4 e LTx5. Experimentos realizados com a toxina recombinante LTx2, em c?lulas musculares BC3H1, mostraram que esse pept?deo bloqueia canais de c?lcio tipo-L e inibe a recarga de Ca2+ dos estoques intracelulares. A estrutura prim?ria do pept?deo LTx2 ? muito similar a dos pept?deos LTx1 e LTx3, portanto, espera-se que essas toxinas possuam mescanismos de a??o semelhantes. Por outro lado, a sequ?ncia de amino?cidos da LTx4 e da LTx5 s?o bem diferentes, tanto quando comparadas com a LTx1, LTx2 e LTx3, quanto entre si, o que leva a acreditar que elas possam atuar em diferentes canais e/ou ter diferentes mecanismos de a??o. Neste trabalho, a sequ?ncia codificante para o pept?deo maduro LTx4 foi subclonada no vetor de express?o pET21b (Novagen). A estrat?gia foi inserir o gene de interesse sob o controle do promotor T7 e obter um produto expresso em fus?o com uma cauda de seis histidinas. A confirma??o da clonagem foi realizada atrav?s de PCR, digest?o do DNA plasmidial com endonucleases de restri??o e, finalmente, seq?enciamento dos DNAs extra?dos dos clones positivos. A indu??o da express?o do pept?deo LTx4 foi realizada com a adi??o de 1mM de IPTG ao meio de cultura. Nos ensaios de imunodetec??o utilizamos o anticorpo monclonal anti His-Tag. O pept?deo recombinante foi identificado tanto em experimentos de Western Blot como em experimentos de Dot Blot. O pept?deo recombinante foi expresso exclusivamente na forma de corpos de inclus?o e em maiores quantidades ap?s tr?s a quatro horas de indu??o. O perfil prot?ico foi analisado em g?is de poliacrilamida (Tricina-SDS). Experimentos de purifica??o do pept?deo encontramse em andamento.Peptide neurotoxins found in animal venoms have attracted great interest of researchers. The possibility of using them as tools for molecular studies of ion channels in addition to use as biopesticides and/or as biopharmaceuticals has boosted research in this area. Experiments with the venom of the spider Lasiodora sp showed that it acts on ion channels of small vertebrates and invertebrates. Recently, we constructed a cDNA library from mRNAs present in the venom gland of this spider. The screen of this library, using ELISA and DNA sequencing, allowed the identification of five toxins in the venom, named LTx1, LTx2, LTx3, LTx4 and LTx5. Experiments conducted with recombinant toxin LTx2 in muscle cells BC3H1 showed that this peptide blocks L-type calcium channels and inhibits Ca+2 recharge from intracellular stores. The primary structure of the peptide LTx2 is very similar to the peptides LTx1 and LTx3, and consequently it is expected that these toxins have similar action mechanisms. On the other hand, the amino acids sequences of LTx4 and LTx5 are quite different, both when compared with LTx1, LTx2 LTx3 and as between themselves, which leads to believe that they can act on different channels and/or have different action mechanisms. In this work, the coding sequence for the mature peptide LTx4 was subcloned at the expression vector pET21b (Novagen). The strategy used was to insert the gene of interest under the control of T7 promoter and obtain the expression product in fusion with a six histidine tag. The cloning confirmation was performed by PCR, plasmid DNA digestion with restriction enzymes, and finally, sequencing of DNAs extracted from the positive clones. The induction of LTx4 peptide expression was performed by addition of 1mM IPTG to the culture medium. At the immunodetection assay, we used monoclonal antibody anti His-tag. The recombinant peptide was identified by both Western Blot and Dot Blot experiments. The recombinant peptide was expressed exclusively as inclusion bodies and in larger amounts after three to four hours of induction. The protein profile was analyzed on polyacrylamide gel electrophoresis (Tricine-SDS). Peptide purification experiments are in course