Attempts to Amplify EDTA Monooxygenase A for Cloning into the pET-32a Expression Vector

Abstract

One significant problem with bacterial overexpression systems is the production of inactive protein in the form of inclusion bodies. The Novagen pET-32a vector was developed as an expression system for increasing the solubility of overexpressed proteins. This system works by fusing Escherichia coli thioredoxin with an insoluble protein of choice. The goal of this study was to compare the effectiveness of thioredoxin and another E. coli redox protein, NADH:flavin oxidoreductase, in increasing the solubility of the highly insoluble protein EDTA monooxygenase A. A strategy for amplifying the EDTA monooxygenase A sequence from another vector pEmoA for its insertion into pET-32a was developed in which mutagenic primers were designed to introduce EcoRI recognition sites flanking the gene to facilitate the cloning process. Several attempts were made to amplify the fragment by PCR in which different experimental conditions were varied, but due to time constraints, the correct PCR product was not obtained. Further work must be done to optimize the PCR for the amplification of the EDTA monooxygenase A gene and to generate the constructs necessary to compare the effectiveness of thioredoxin and flavin oxidoreductase in increasing protein solubility.