The Production of an Antibody Against the Alpha Subunit of the Glucosidase II Enzyme

O. UZOMAH, C.A. LABRIOLA, A.J. PARODI, AND C. D’ALESSIO.

San Diego, California, USA

Workshop; Experimental Biology Conference; 2007

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.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.