LOW COST EXTRACTION AND PURIFICATION OF AVIDIN FOR ITS USE AS A DIAGNOSTIC REAGENT

PAMELA KIKOT; MIRNA AGUILERA; MARIANO GRASSELLI

Otro; REUNIÓN CONJUNTA SAIB-SAMIGE; 2020

SAIB SAMIGE

Avidin is a glycoprotein of approximately 68 kDa, which is composed of four identical subunits. It is a basic protein,very soluble in aqueous solutions and with a great stability at wide ranges of pH and temperature. The high affinity forBiotin has turned Avidin into a very important tool for a numerous of biochemical assays and affinity based compoundseparations. Nowadays, its commercial cost is in the order of 40 U$S /mg.Although Avidin is obtained from chicken egg white, an accessible raw material, its extraction with high purity andefficiency represents a real challenge because it is in a very low quantity (less tan 1 mg/egg) and concentration (lessthan 0,03% of total proteins in egg white).In this work, the extraction of Avidin from egg white using chromatographic systems was studied. In a first ionexchange chromatography step, the basic proteins were recovered, Lysozyme and Avidin mainly. A high capacitysulfonic matrix based on a composite of cotton fibers was used, which was produced in our laboratory. This matrixallows the process of large volumes of egg white without a filtration or clarification step. This chromatography bed hasvery good mechanical properties and low cost. Therefore, in case of irreversible plugging it can be discarded.Most protein contaminants were eliminated during the ion exchange chromatography step. As a second and last step ofpurification, an affinity chromatography was optimized using a iminobiotinylated matrix, from which 0.3 mgAvidin/egg was recovered. Thus, it was possible to obtain Avidin with a purity of more than 95%, which is functionallycomparable with commercial Avidin for its use in ELISA and other immunoassays.