MOLECULAR CLONING, EXPRESSION AND PURIFICATION OF A ANTIMICROBIAL TOXIN FROM THE VENOM OF THE Lasiodora sp. SPIDER

DUTRA A.A.A.(1), SOUSA L.O.(1), KALAPOTHAKIS E. (2), CASTRO I.M.(1)

(1) Laboratório de Biologia Celular e Molecular, Núcleo de Pesquisa em Biologia – NUPEB – Universidade Federal de Ouro Preto, (2) Departamento de Farmacologia, ICB – Universidade Federal de Minas Gerais.

Venom from snakes, scorpions, spiders and other venomous animals is a rich source of potent toxins. An interesting feature of some toxins is their remarkable species specificity. In the present work we cloned a sequence which codifies for the toxin LTx2 from the venom of the spider Lasiodora sp, in the expression vector pET11a. This toxin was previously identified in the screening of the cDNA library of the total venom. After cloning in expression vector we verified if the fragment was inserted in the correct frame using the Sanger DNA sequencing method. The expression of the target protein was made by adding 0,6 mM IPTG to the media followed by a 3 to 4 hours incubation. With the assistance of immunochemical assays we were detected that the protein was been expressed in the form of inclusion bodies. Then we defined a strategy to recover the soluble refolded protein. Using a solution containing 6 M urea we proceed the solubilization of the inclusion bodies. The refolding of the soluble protein was made by the dialysis method in refolding buffer. The sample was, then, submitted to HPLC, in a reversed phase column, to purify the protein. The purifying process was efficient, as shown by the Western Blotting and electrophoresis results. After that, we made an anti microbial assay, where we could see that the target protein, in the concentration of 400 mg/mL, inhibited the growth of the organisms Escherichia coli TOP 10F’, Salmonella agona and Staphilococcus epidermidis. The expression and recovery of larger amounts of the target protein will allow tests with this toxin in other organisms and electrophysiological experiments.

KEY WORDS: Spider, Lasiodora sp, cloning, purification, expression, antimicrobial, toxin, venom.

FINANCIAL SUPPORT: FAPEMIG, CNPq and UFOP

CORRESPONDENCE TO: I. M. Castro, LBCM, NUPEB – ICEB-II, UFOP. Campus Morro do Cruzeiro, Ouro Preto, MG. Brazil. CEP: 35400 – 000.