PCR Amplification of emoA from the pEmoA Plasmid

Abstract

Bacterial expression systems, such as the Novogen pET32a(+) vector, are frequently used to increase the solubility of recombinant proteins and circumvent inclusion body formation in Escherichia coli by expressing the desired products as fusion constructs with highly soluble proteins such as E.coli thioredoxin A. In this study we amplified emoA, from the plasmid pEmoA, using the polymerase chain reaction, in order to study the potential role of flavin oxidoreductase on solubility of EDTA monooxygenase A (emoA). PCR reactions were conducted using custom primers designed on the putative emo A sequence from GenBank. Initial reactions showed no amplification of the target even though the controls were amplified. Annealing temperature, polymerase enzyme, and DNA concentration were varied, along with the use of bovine serum albumin and dimethylsulfoxide as PCR enhancers. Restriction digest analysis was performed to confirm the identity of the putative emo A gene in the pEmoA plasmid and Mfold analysis was performed in order to assess the potential secondary structure of pEmoA. The Mfold analysis revealed a significant hairpin in the sequence region adjacent to the reverse primer. Amplification of the target sequence was finally achieved with the addition of 5% v/v dimethylsulfoxide that would have melted the predicted hairpin.