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Abstract
The formation of inclusion bodies is a major obstacle for getting efficient bacterial protein production in expression systems. The pET32a(+) expression vector from Novagen fuses protein of interest to thioredoxin A (TrxA) to help facilitate disulfide bond formation in the cytoplasm greatly reducing protein aggregates. Flavin oxidoreductase (Fre) is another thioredoxin enzyme and may present another option. An attempt was made to clone the PCR amplified fre gene into pET32a(+) that had the thioredoxin A (TrxA) tag removed. Since it is unknown how the redox state of thioredoxin enzymes are able to improve protein solubility we wanted to compare expression of the highly insoluble protein EDTA monooxygenase A (EmoA) as a fusion protein to Fre and as independent coexpression. The project was delayed at the cloning step due to the difficulty of working with the NdeI restriction enzyme. It was found that a ligation condition with a low amount of T4 DNA ligase improve the poor ligation efficiency of NdeI digested fragments. A recircularized vector with the TrxA tag removed was made as well as three potential clones that may contain the Fre insert. Attempts to clone EDTA monooxygenase A into the new vector resulted in no clones.