IN VITRO AND IN VIVO CHAPERONE ACTIVITY OF THE PHASIN PhaP FROM Azotobacter sp. FA8

MARIELA P. MEZZINA; WETZLER, D. E; DINJANSKI, N,; PRIETO, M, A; M. JULIA PETTINARI

Córdoba

Congreso; XI Congreso Argentino de Microbiología General (SAMIGE); 2015

Sociedad Argentina de Microbiología General (SAMIGE)

Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. PhaP from Azotobacter sp. FA8 (PhaPAz ) is a representative of the prevailing type in the multifunctional phasin protein family. This protein has been cloned and expressed in Escherichia coli. Recombinant E. coli strains overexpressing phaPAz grow more, and accumulate more polymer, suggesting that PhaPAz exerts a growth promoting effect. Expression of PhaPAz was also observed to have an unexpected protective effect in E. coli strains that do not synthesize PHAs, under both normal and stress conditions, resulting in increased growth and higher resistance to both superoxide stress and heat shock. In vitro chaperone activity experiments were performed in order to shed light on the mechanisms by which PhaPAz exerts its protective effect. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate sinthase (CS) and to facilitate the refolding process of the enzyme after chemical denaturation. These experiments showed that PhaPAz presents in vitro chaperone activity. Fluorescence microscopy and electron transmission microscopy were used to study the role of PhaPAz in in vivo protein folding, protein aggregation and inclusion body dynamics. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that forms large inclusion bodies when overexpressed. A reduction in the number of inclusion bodies of PD was observed when the insoluble protein PD was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural PHA producers, that in these microorganisms is masked by the fitness enhancing properties of the polymer. These observations open the door for novel biotechnological applications of this protein, for example, in the production of recombinant proteins and other heterologous products in E. coli.