Cloning EDTA monooxygenase as a Model Protein to Characterize the Effects of Flavin oxidoreductase on Solubility of Proteins in Protein Overexpression Systems

Abstract

Escherichia coli thiol reductase has been utilized by Novagen to create an expression system for insoluble and poorly soluble proteins which normally form aggregates when overexpressed and become inactivated. Believing that the redox state maybe responsible for the solubilizing mechanism, we conducted the following study to assess the role of E. coli flavin oxidoreductase in increasing solubility of such proteins. Primers were designed for amplification of E. coli flavin oxidoreductase. We attempted replacing the thiol reductase sequence in the Novagen vector with the amplified sequence. Due to time limitations, we were only able to construct a control vector with an insoluble protein sequence, EDTA monooxygenase A. Construction of other vectors were attempted but failed due to specific properties of restriction enzymes used. Further work is required to complete the constructs necessary to fully examine the effects of flavin oxidoreductase on protein solubility.