Purification of the basic protein avidin using Gradiflow technology

• Published in: Protein expression and purification Vol. 26; no. 1; pp. 149 - 152
• Main Authors: Rothemund, Deborah L, Thomas, Theresa M, Rylatt, Dennis B
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2002
• Subjects: Animals; Avidin - isolation & purification; Chickens; Egg White; Electrophoresis - instrumentation; Electrophoresis - methods; Hydrogen-Ion Concentration; Muramidase - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/S1046-5928(02)00517-X

The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, p
I 10, from chicken egg white. Using a charge based separation at pH 9.0, the high p
I of avidin and lysozyme (p
I 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5
h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60–65% from one egg white with minimal lysozyme activity detected.