Soluble expression of horseradish peroxidase in Escherichia coli and its facile activation

https://doi.org/10.1016/j.jbiosc.2018.04.004Get rights and content

Horseradish peroxidase (HRP) is widely used as a marker enzyme in immunoassays and biosensors, and can possibly be used in industries such as waste water treatments or fine chemical synthesis. Cost-effective production of active HRP is thus very important in the related fields. Also, engineering of HRP for its better performance in the designated application is expected to make the enzyme even more important in several areas of research and industry. One of obstacles to this end and to the large scale production of the enzyme has been its facile expression in a bacterial host. Here we show that HRP could be overexpressed as a soluble form by fusing the enzyme with Escherichia coli phosphoglycerate kinase (PGK). After simple incubation with calcium ion, hemin, and oxidized glutathione, PGK-HRP could be fully activated showing a higher molar specific activity than plant-derived HRP. Our established procedure did not use tedious and inefficient refolding steps that have been used to activate HRP produced as inclusion bodies and thus is superior in its overall yield (>72 mg purified HRP conjugate per L culture) to existing methods. By co-expressing PGK-HRP with ferrochelatase in a special host that permitted the formation of disulfide bonds in the cytoplasm, the activation steps could be simplified even though the resulting specific activity was low.

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Materials

All the chemicals and plant-derived HRP (Type II) were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise stated. Kits for cloning and DNA preparations were from Enzynomics (Daejeon, Korea) and CosmoGenetech (Seoul, Korea), respectively. Primers for the polymerase chain reactions were synthesized from CosmoGenetech.

Vector construction

The nucleotide sequences of primers and genes used in this study are presented as Supplementary information accompanying this paper. For the construction of

Soluble expression of HRP

Since refolding of HRP followed by the activation with heme and Ca2+ had been demonstrated (21), it seemed plausible that similar activation would be possible once the soluble expression of HRP was achieved. Directing synthesized HRP to the periplasm of E. coli for proper disulfide formation and thus for soluble expression could be one way to achieve the goal. It was shown that partially active HRP could be expressed in the periplasm of E. coli 21, 22. An independent study with a similar

Discussion

Here we successfully demonstrated a new strategy to produce active HRP in E. coli. Using the solubility-enhancing fusion partner, the target protein was overexpressed in a soluble form. Subsequent activation with Ca2+, hemin, and GSSG afforded the molar specific activity comparable or better than the plant-derived, commercial HRP. We routinely recovered more than 7.2 mg of purified PGK-HRP from 100 mL of culture, which could be improved by employing a bioreactor with optimized feeding

Acknowledgments

This work was supported by National Research Foundation of Korea (NRF-2017R1D1A1B03033679).

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