The production of an antibody against the alpha subunit of the Glucosidase II enzyme

ONYEKA N. UZOMAH; CECILIA D'ALESSIO; ARMANDO PARODI

Kansas City, Missouri, USA

Congreso; SACNAS (Society for the Advancement of Chicanos and Native Americans in Science); 2007

Society for the Advancement of Chicanos and Native Americans in Science

The Production of an Antibody Against the Alpha Subunit of the Glucosidase II Enzyme 1Onyeka N. Uzomah, 2,3Cecilia D´Alessio and  2Armando Parodi 1Biology, CSUDH; 2Argentina Fundación Instituto Leloir and IIBBA, CONICET, Buenos Aires, Argentina; 3FBMC, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina   Glucosidase II (GII) plays a very important role in the mechanism of N-glycan quality control of glycoprotein folding in the endoplasmic reticulum (ER). Genetic evidence in previous studies has shown that the GII structure is heterodimeric, the GII-alpha homolog responsible for catalytic activity, and GII-beta 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 location of GII-alpha in the cell structure of Schizosaccharomyces pombe beta minus. A PCR reaction using the S. pombe genomic DNA as template generated a fragment of  about 1000 base pairs. The pET system vector was used to. transform E. coli BL26 strain. The transformed bacteria were induced to express the recombinant protein which was used to produce the above mentioned polyclonal antibodies. A protein of the desired weight of about 35kDa was obtained at a concentration of 0.7 mg/ml. The protein has been injected into a rabbit to produce the antibodies. 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.