Production of recombinant avidins in Escherichia coli and insect cells

Abstract

Avidin (Avd) is a basic glycoprotein which constitutes about 0.05% of the protein in the chicken egg-white. Due to the extraordinarily strong binding characteristics between Avd and biotin, Avd is widely used tool in numerous applications of (strept)avidin-biotin technology. In order to further expand this technology and to be able to remove the unwanted intrinsic properties of Avd (the high pI and the sugar moieties) by genetic engineering, this study focused on the establishment of a production system for recombinant Avd (re-Avd) and Avd fusion proteins. Our initial attempts to produce re-Avd in E. coli resulted in low amounts of protein. However, by adapting the first ten codons of re-Avd to the so-called optimal E.coli codon usage, we were able to increase the production of re-Avd at 37°C to over one milligram per liter of culture. However, at 37°C most of the re-Avd was in insoluble form. At 25°C the protein was mostly soluble, but the overall yield of re-Avd was significantly reduced. The production of Avd as Nor C-terminally linked to glutathione S-transferase (GST) followed the same pattern, but significantly larger amounts of fusion proteins were synthesized at both temperatures. The position of the Avd-tag in the fusion protein has no affect on either the biological activity of re-Avd or that of GST. Biologically active re-Avd and its fusion proteins were found to be easily purified in a single step. The baculovirus expression vector system proved to be more efficient tool for the synthesis of avidins (Avd and its derivatives) in milligram amounts than E.coli. The avidins were obtained in both secreted and intracellular forms from insect cells in a single step. The purified avidins were glycosylated and assembled mainly in tetramers. Like native avidin, the recombinant tetramer also exhibited a high level of thermostability, and was further stabilized upon binding biotin.