Protein Engineering of Avidin by Rational Design & DNA Family Shuffling

Acta Universitatis Tamperensis No. 1901


By: Barbara Taskinen
March 2014
Tampere University Press
Distributed by Coronet Books
ISBN: 9789514493591
165 Pages
$97.50 Paper original


Evolution is caused by mutations in the DNA of organisms. Mutations change the properties of proteins, which are machines that carry out the functions necessary for life. Mutations have either a positive or negative effect on the survival and propagation of organisms and are submitted to natural selection. Evolution can be mimicked in the laboratory by directed or random mutagenesis of the DNA that encodes for a protein. In directed mutation one specifically selected building block of the protein is changed into another, whereas in random mutagenesis the changes are made randomly.

One example for random mutagenesis is DNA family shuffling, where the DNA of different organisms encoding for the same protein is cut into small pieces and reassembled into DNA that contains parts originated from different organisms. This creates a high amount of different variants from which the variant with the desired target function can be selected by using a method called phage display, which is a laboratory version of natural selection. In phage display, the mutated protein is displayed on the surface of a bacterial virus called phage. The phage contains the DNA encoding for the protein expressed (displayed) on the phage surface, and thus the function of the protein is connected with the DNA encoding it.

We used directed mutagenesis and DNA family shuffling to alter the function of the protein avidin. Avidin is a highly stable protein that tightly binds to the small molecule biotin. It can be found in a variety of organisms including vertebrates, fungi and bacteria. Its function and structural properties makes it a good candidate to develop life sciences applications where immobilization of molecules is required. We were able to alter the function and structural properties of avidin using DNA family shuffling in combination with phage display and by applying directed mutagenesis. In addition we characterized avidin from zebrafish.