Expressão recombinante da canacistatina em células de inseto
Resumen
Due to the great economic importance of sugarcane crop in Brazil, the occurrence of
infections by phytopathogens leading to decreased productivity and quality remains a serious
problem. As the plants have natural mechanisms of defense against the attack of fungi
and insects, amongst which the inhibitors of proteases are distinguished, the use of these
substances in the development of resistant plants or in pesticide production may be an interesting
alternative. One of the major classes of protease inhibitors acting against the attack
of pathogens is the cystatins, proteins that inhibit cysteine proteases specifically. Canecystatin
was the first cysteine protease inhibitor characterized from sugarcane. This gene
codes for a ~ 14 kDa protein that contains conserved regions common to the cystatin family.
Although the protein has previously been produced in a bacterial system of expression,
in this work we use this sugarcane cystatin as a model for the implementation of a heterologous
protein expression system in insect cells. This system has some advantages relatively
to the prokaryotic system such as the possibility of posttranslational modifications.
The recombinant protein was expressed in this system in a soluble form and purified using
affinity chromatography in a nickel column, rendering approximately 23 mg/L of pure protein.
The activity of the protein was assayed against papain, being capable of inhibiting the
activity of this cysteine protease efficiently. Furthermore, the stability of the protein was
analyzed in different conditions of pH and temperature. We conclude that the canecystatin
is a good model for the implementation of the Baculovirus Expression System at the Molecular
Biology Laboratory of the UFSCar