Expressão heteróloga, purificação e estudo de atividade de uma proteína inibidora de cisteínio protease da cana-de-açúcar e posterior evolução in vitro pela técnica de DNA Shuffling.

Abstract

Plants seem to have developed, over a long period of time, defence mechanisms against fungi and insects. One of them is the use of protease inhibitor proteins. Cystatins are proteins that inhibit specifically cysteine proteases. They occur naturally in several vegetable species and it is believed that they work in the defence mechanism from plants against some pathogens. The Canecystatin gene which codifies a protein containing 106 amino acids residues, was identified in sugarcane and bears significant similarity with oryzacystatin, a cystatin from rice. The recombinant protein was expressed in E.coli in the soluble form, which promoted its direct purification by affinity chromatography in nickel column. The purified protein was analysed by Circular Dichroism (CD) and displayed estimated secundary structure similar to oryzacystatin I. Besides that, the protein was submitted to crystallization assay, and it was possible to form protein crystal, which will enable future studies by X-Ray Crystallography. Canecystatin, was used successfully in growth inhibition tests against the filamentous fungus Trichoderma reesei. The protein minimum inhibitory dose were about 50 µg/ml. The recombinant canecystatin was also able to inhibit plant pathogenic fungi including Colletotrichum sp P25, Colletotrichum sp P28, Colletotrichum sp P10, Fusarium moniliforme and Aspergillus niger. The most efficient inhibition was obtained against Colletotrichum sp P10 (32.25µg/ml). In order to obtain cystatin with improved activity direct evolution tests were carried out. A shuffling library was constructed using two diferent cystatins, i.e Canecystatin and Oryzacystatin I. One clone formed by this proteins was selected. This clone was expressed, purified and subjected to activity tests, and the results shown that the activity of mutant protein increased, in particular regarding its inhibit activity of Cathepsin B. Our findings open perspectives of using this protein as a natural fungicide and of developing more resistant varieties of sugarcane .