Expressão Recombinante de uma Miraculina de Citrus em Pichia pastoris e em Escherichia coli
Abstract
The high consumption of sugar in Brazil and in the world result in drastic consequences
for human health, causing numerous diseases. In the search for alternatives to reduce
sugar consumption, several studies have emerged related to the development of caloric
and non-caloric sweeteners, of natural or artificial origin. Among the natural compounds
there is a group of proteins that have the property of modifying the flavor of food. Within
this class of molecules, there is the protein called miraculin, from the plant Richardella
dulcifica, native to Africa, which has the property of modifying the acidic taste of food
into sweet. Due to this potential, there are reports of several tests carried out in an attempt
to produce miraculin through recombinant expression in other organisms. However, other
studies have discovered the existence of miraculin-like proteins in other plants, although
there is still no description of miraculin-like proteins with such property. On the other
hand, in silico analyzes allowed finding in Citrus paradisi a protein with a gene sequence
with high similarity to that found in the original miraculin. Therefore, the characterization
of this protein and the exploration of its potential ability to modify flavor may result in
non-caloric alternatives to sweeten acidic foods, thus contributing to an improvement in
human health. Therefore, the objective was the recombinant expression of a miraculin
derived from Citrus paradisi, called Citros_Mir, in Pichia pastoris yeast and in
Escherichia coli bacteria. Cloning was performed in the propagation vector pGEM-T.
Expression in E. coli occur with pET-28a expression vector and the Rosetta strain, in
addition to testing temperatures of 20, 30 and 37ºC, and final concentrations of 0.2; 0.4
and 1.0 mM of the IPTG expression inducer. Expression in P. pastoris was performed
with the pPICZαC expression vector and the X-33 and KM71H strains. In this system,
was test the final concentrations 0.75, 1, 2 and 3% of the methanol expression inducer,
concluding that the yeast presents a better expression of proteins in a culture medium
containing 2% of methanol. The results obtained showed that attempts to express
Citros_Mir in these systems were not efficient, suggesting the need for new tests,
modifying conditions and/or expression systems.
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