INVESTIGADORES
FIDELIO gerardo Daniel
congresos y reuniones científicas
Título:
Viper Venom phsopholipase A2: Heterologous production and renaturation
Autor/es:
YUNES QUARTINO P.J.; FIDELIO G.D.
Lugar:
Salta
Reunión:
Congreso; Latin American Protein Society Meeting and XXXIX Argentinean Biophysical Society (SAB); 2010
Institución organizadora:
Latin American Protein Society Meeting and XXXIX Argentinean Biophysical Society (SAB)
Resumen:
We have previously reported (SAB meeting,
2008) the first cloning of isoenzymes of phospholipase A2 (PLA2)
from Bothrops diporus (Yarará chica),
a common viper of Argentina, and the expression of one of these in inclusion
bodies fused to a N-terminal 6xhistidine-tag and a thrombin recognition sequence.
Hydrolysis of dLPC monolayers upon addition of that construction was not
observed. This, along with some reports that indicate the importance of a
native N-terminal sequence to obtain full interfacial catalysis1,
prompted us to set up a system to obtain native sequence protein.
Expression of one of these
isoenzymes fused to an ubiquitin moiety was successfully achieved. Fusion protein
was found in inclusion bodies. Its reduction and complete denaturation were
carried out to solubilize the Ubiquitin-PLA2 protein. After this, solution was dialyzed
against oxidation buffer, followed by a ten-fold dilution. To cleave off
Ubiquitin and obtain PLA2 with native N-terminus, peptidase USP2cc2
was added. After this, PLA2 activity was detected as hydrolysis of
monolayers of dLPC (substrate), followed by the surface barostat technique and
the optimal dLPC lateral pressure for hydrolysis was determined3. No
PLA2 activity could be detected prior the addition of USP2cc.
To our knowledge this is the first
report of heterologous production of an active phospholipase A2 from
Bothrops diporus and utilization of the
Ubiquitin/USP2cc system to obtain any PLA2 in E. coli.