Clonagem da superóxido dismutase de Chromobacterium violaceum e expressão heteróloga

Abstract

The main objective of this work was to isolate the ORFs CV0867 (sodB2) e CV2504 (sodB1) of C. violaceum and induce their expression in Escherichia coli cells harboring sod genes, to evaluate the increase in SOD activity in the recombinant cells exposed to oxidative stress conditions. The sodB1 gene was isolated by PCR, using specific primers, and cloned into pGEM ® -T Easy vector. The amplification reaction could not be optimized for the second set of primers, specific for sodB2 gene. In an attempt to obtain a vector suitable for the regulated heterologous expression of SOD, some strategies were proposed for the construction of vectors with expression under the control of a sterically repressed promoter – srp. The sodB1 gene was then subcloned into vectors pCR4-SRP and pUC-SRP, derived from pCR ® 4-TOPO ® e pUC72, respectively, both developed during this project. However, the obtaining of recombinant clones was not possible. Despite the fact that SOD is not a toxic protein for the cell, we tested the hypothesis of the lethality of recombinant clones caused by the overexpression of SOD. Thus, amy and pfu genes were cloned into pUC-SRP, although the clones harboring the gene of interest could not be obtained. We concluded that the expression mechanisms of pUC-SRP and pCR4-SRP vectors may have caused deletery effects to the host cells, and that modifications in their structure should be made for the minimizing of heterologous expression levels, reducing the risk of toxicity for the clones.