The Synthesis of an Antibody against the Alpha Subunit of the Glucosidase II enzyme of S. pombe

ONYEKA N. UZOMAH; CARLOS LABRIOLA; ARMANDO J. PARODI; CECILIA D'ALESSIO

San Diego, California, USA

Conferencia; Federation of American Societies for Experimental Biology (FASEB) Conference; 2008

Federation of American Societies for Experimental Biology (FASEB)

Glucosidase II (GII) plays a very important role in the mechanism of glycoprotein foldingquality control in the endoplasmic reticulum (ER). Genetic evidence in previous studieshas shown that the GII structure is heterodimeric; the GII-alpha subunit is responsible forcatalytic activity, and GII-beta subunit for retaining the enzyme in the ER. The purpose ofthis work is to create a specific antibody that can be used to detect the subcellular locationof the GII-alpha subunit in the fission yeast Schizosaccharomyces pombe. A PCRreaction using the S. pombe genomic DNA as template and primers corresponding to aportion of GII-alpha encoding gene generated a fragment of about 1000 base pairs thatwas cloned in the pET system vector. The transformed E. coli BL26 strain was theninduced to express the recombinant protein. The His-tagged protein was affinity purifiedand used to produce the above mentioned polyclonal antibodies. The protein was injectedinto a rabbit to produce polyclonal antibodies. The recombinant protein was recognized inthe serum of the animal, but not in the total cell extract of S. pombe. More booster shotswill be given to the rabbit to increase the protein levels in the serum. It is hoped that thisantibody will play a significant role in further experiments to confirm whether the betasubunit is necessary for retaining the alpha subunit of GII in the ER.