Low cost extraction and purification of Avidin for its use as a diagnostic reagent

KIKOT, P.A.; AGUILERA, MIRNA; MARIANO, GRASSELLI

CABA

Congreso; REUNIÓN CONJUNTA SAIB-SAMIGE; 2020

SAIB

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 for Biotin has turned Avidin into a very important tool for a numerous of biochemical assays and affinity based compound separations. 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 and efficiency represents a real challenge because it is in a very low quantity (less tan 1 mg/egg) and concentration (less than 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 ion exchange chromatography step, the basic proteins are recovered, Lisozyme and Avidin mainly. A high capacity sulfonic matrix based on cotton fibers was used, which was produced in our laboratory [1]. This matrix allows the process of great volumes of egg white without needing a filtration or clarification step since it works not only as an ion exchanger but also as a filter itself. This chromatography bed has very good mechanical properties and in case of plugging, it can be discarded because of its low costs [2]. The most part of protein contaminants are eliminated when carrying out the ion exchange cromatography step. As a second and last step of purification, an affinity cromatography was optimized using a biotinylated resin from which 0,3 mg Avidin/egg was obtained. Thus, it was possible to obtain Avidin with a purity of more than 95%, which is functionally comparable with commercial Avidin for its use in ELISA and other immunoassays. References [1] Fernandez-Lahore M, Grasselli M, inventors; Jacobs University gGmbh/UNQ, assignee. Composite material. United States patent application US 13/681,427. 2013 May 9.[2] Gavara, P.R., Cabrera, R., Vennapusa, R.R., Grasselli, M. and Fernandez-Lahore, M., 2012. Preparation, characterization, and process performance of composite fibrous adsorbents as cation exchangers for high throughput and high capacity bioseparations. Journal of Chromatography B, 903, pp.14-22.