Arginine-kinase of spider polybetes pythagoricus: cdna description and the obtaining of recombinant protein

LAINO ALDANA; CARRASCO SALVADOR; GARCIA-OROZCO KARINA; LOPEZ-ZAVALA ALONSO; CUNNINGHAM MÓNICA; ROGERIO SOTELO; GARCIA FERNANDO

Mar del Plata

Congreso; LI Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular.; 2015

Sociedad Argentina de Bioquímica y Biología molecular

Phosphagens in invertebrates are important because of their bioenergetic function, and as allergens for human beings. Particularly in spiders, studies are scarce. The arginine kinase enzyme (AK, EC 2.7.3.3) maintains the levels of phospho-arginine phosphagen by the reversible phosphorylation of L-arginine with ATP. In the present work it was identified the coding region of AK gen of the spider Polybetes pythagoricus (Subphylum Chelicerata, Class Arachnid) performing a cDNA library through reverse transcription of mRNA of muscle tissue to obtain data on expressed genes. Through Sanger sequencing and assembling a cDNA fragment was obtained for AK and its sequencing was completed through the technique of rapid amplification of cDNA ends. It could be determined that it shows 384 amino acids, a theoretical molecular mass of 43 kDa, and an isoelectric theoretical point of 6.37. From the bioinformatics analysis it was observed that it shows 93% of identity with AK of the spider S. mimosarum and 81% of identity with AK of horseshoe crab, considered as the phylogenetically basal chelicerate L. polyphemus (Subphylum Chelicerata, Class Merostomata). By means of the construction of a synthetic gen it was possible to express the protein recombinant in E. coli. These studies will enable us to perform a biochemical, biophysical, and structural characterization.