Cloning and Expression of a Fibrin(ogen)olytic Serine Protease from Solanum tuberosum.

PEPE, A.; WHITEHEART, SYDNEY W,; MARÍA G. GUEVARA.

Estados Unidos de América. Memphis, TN.

Conferencia; 16th Midwest Platelet Conference; 2016

The University of Tennessee

Gene cloning and expression of a serine protease from Solanum tuberosum with fibrin(ogen)olytic activityAuthors and affiliations (foni, acá cuando pongas el autor de contacto ponenos tanto a mi como a wally)IntroductionPlant serine proteases are enzymes widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and anti-platelet agents. Previously, in our lab we have isolated and characterized a serine protease named as StSBTc-3 from Solanum tuberosum. StSBTc-3 is an enzyme with potential therapeutic applications because it has fibrinogenolytic activity. In this work we present the molecular cloning and hetelogous expression of StSBTc-3 Methods and resultsDNA was extracted from Solanum tuberosum tubers. Polymerase chain reactions (PCR) where carried out using gene-specific oligonucleotide primers and potato DNA as template. The fragment corresponding to the peptidase domain of the StSBTc-3 genes was amplified. The PCR product was cloned into pPROEX HTb vector and sequenced.E. coli Rosetta Competent Cells were transformed and the expression was induced with 1mM of IPTG for 4 hs. The cells were harvested and the enzyme was purified.The cloning procedure was successful and presence of the insert into the plasmid was confirmed through DNA sequencing. The mature gene expressed low soluble StSBTc-3 activity and large amount of insoluble protein in inclusion bodies without enzyme activity. The recombinant cells were lysed with 0.1 mg/ml lysozyme and ultrasonic shake. After centrifugation the pellet was washed twice and dissolved with 8M urea. The recombinant protein containing His-tag was purified with a Ni-NTA column.A rapid refolding protocol was tested mixing directly the protein solution with buffer 50 mM Tris/HCl pH 8 in a 1:10 ratio. The resulting solution was dialyzed against the same buffer to eliminate the rests of urea and concentrated. The fibrinogenolytic activity was evaluated incubating the previous solution with different concentration of human fibrinogen.The refolded recombinant StSBTc-3 was not active in the conditions tested.ConclusionsLarge amount of expressed StSBTc-3 was present in inclusion bodies. The recombinant enzyme could be dissolved with the 8M urea solution. The enzyme solution resulting after the refolding process didn?t show fibrinogenolytic activity. Different strategies will be applied in the future, as different refolding solutions or a different cloning vector to avoid the precipitation of the enzyme into de inclusion bodies. Acá yo agregaría conectores ya que de lo contrario, son diferntes conclusions.