IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The Production of an Antibody Against the Alpha Subunit of the Glucosidase II Enzyme
Autor/es:
ONYEKA N. UZOMAH; CARLOS LABRIOLA; ARMANDO PARODI; CECILIA D'ALESSIO
Lugar:
Carson, California, USA
Reunión:
Jornada; Student Research Day at California State University, Dominguez Hills; 2007
Institución organizadora:
California State University, Dominguez Hills
Resumen:
The Production of an Antibody Against the
Alpha Subunit of the Glucosidase II Enzyme
Authors: 1Onyeka N.
Uzomah, 2Carlos Labriola, 2Armando
Parodi, 2, 3Cecilia D'Alessio
Departments: 1Biology, CSUDH; 2Argentina Fundación
Instituto Leloir and IIBBA, CONICET, Buenos Aires, Argentina; 3FBMC,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Argentina
INTRODUCTION: Glucosidase II (GII) plays a very important role
in the mechanism of glycoprotein folding quality control in the endoplasmic
reticulum (ER). Genetic evidence in previous studies has shown that the GII
structure is heterodimeric, the GII-alpha subunit is responsible for catalytic
activity, and GII-beta subunit is responsible for retaining the enzyme in the
ER. The purpose of this work is to create a specific antibody that can be used
to detect the subcellular location of the GII-alpha subunit in the fission
yeast Schizosaccharomyces pombe.
METHODS: A PCR reaction using the S. pombe genomic DNA as template and primers corresponding to a
portion of GII-alpha encoding gene generated a fragment of about 1000
base pairs that was cloned in the pET system vector. The transformed E. coli
BL26 strain was then induced to express the recombinant protein. The His-tagged
protein was affinity purified and used to produce the above mentioned
polyclonal antibodies.
RESULTS: A protein of the desired weight of about 35kDa was
obtained. The protein was injected into a rabbit to produce polyclonal
antibodies. The recombinant protein was recognized by the serum of the animal, and
also in the microsomal fraction of S.
pombe.
DISCUSSION: It is hoped
that this antibody will play a significant role in further experiments to
confirm whether the beta subunit is necessary for retaining the alpha subunit
of GII in the ER.