Development of a rapid and high throughput yeast-based system for expression and purification of AID

Borzooee, Faezeh (2017) Development of a rapid and high throughput yeast-based system for expression and purification of AID. Masters thesis, Memorial University of Newfoundland.

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Abstract

The DNA mutator enzyme, activation–induced cytidine deaminase (AID) is expressed in mature B lymphocytes, and initiates the secondary antibody diversification events of somatic hypermutation (SHM) and class switch recombination (CSR) which mutates cytidine to uridine at Immunoglobulin loci. To date, the expression hosts for purification of AID/APOBECs include bacteria, insect or mammalian cell lines. Here we established and optimized a novel yeast–based expression/secretion system to express AID. This is compatible with different purification tags including GST and His positioned at either N- or C-termini or non-tag. This method also can be adapted for protein secretion into the media, allowing for a simpler purification method of concentrating and collecting the protein from the media, rather than current conventional purification methods. As proof of principle, we provide the first report of expression of functionally active untagged AID as well as GST-AID and AID-His which can address several important unanswered questions remaining in the field.

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