Difference between revisions of "Part:BBa K3037000"

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pOCC97 plasmid backbone for expression (optimized)

Horseradish Peroxidase (HRP)

The TU Dresden 2019 team design this biobrick in order to make a fusion protein with dCas9 in accordance to the RFC 25 standard. (more information)

In the experiment, HRP was used as a color proof reading for detection of specific DNA sequences.

HRP was inserted into the pOCC97 vector for transformation and expressed in E. coli .

Biology

HRP is an extensively studied and one of the most important enzymes obtained from plants. The reason because of the interest in the enzyme is because it has a lot of commercial and practical applications (Veitch, N. C. 2004).

HRP is a peroxidase which oxidizes different substrates (e.g. aromatic phenols) using commonly H2O2, as initial electron acceptors. Physiologically, HRP is involved in many reactions, such as the regulation of the levels of H2O2 and the crosslinking of phenolic molecules. Because of its large amount of different functions, HRP has many isoenzymes. (Krainer, F. W. et al 2014)

The oxidative properties of the HRP allow them to produce color changes in specific substrates. Therefore, in the industry HRP has many applications, especially biosensors and diagnostic kits (e.g., immunoassays, ELISA, EMSA…). (Krainer, F. W. et al 2014)

Also, HRP has many characteristics that make it suitable for therapeutic use as it is stable at 37 °C, shows high activity at physiological pH and can be conjugated to antibodies or lectins. (Humer, D., & Spadiut, O. 2019) In addition, site-directed mutagenesis and directed evolution techniques are beeing used to improve the properties of the HRP. (Veitch, N. C. 2004). The commercially available HRP is extracted from Armoracia rusticana roots. However, Armoracia rustica require long cultivation times and produce low yields which make the classical production method quite inefficient. (Humer, D., & Spadiut, O. 2019)

As a consequence, many studies have addressed Saccharomyces cerevisiae or Pichia pastoris as host organisms. However, these organisms have problems to produce glycoproteins with disulphide bridges. In contrast, E. coli has shown to have no obstacles due to hyper-glycosylation and it is also a suitable organism because of its cheap and easy cultivation. (Humer, D., & Spadiut, O. 2019)

Sequence

pOCC97 plasmid backbone for expression (optimized)

Design Notes

Followed by the biobrick prefix there is an RBS with an NgoIV restriction site, which is used for the RFC 25 standard when trying to make fusion proteins.

References

Veitch, N. C. (2004). Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry, 65(3), 249-259.

Krainer, F. W., Pletzenauer, R., Rossetti, L., Herwig, C., Glieder, A., & Spadiut, O. (2014). Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris. Protein expression and purification, 95, 104-112.

Humer, D., & Spadiut, O. (2019). Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis. International journal of molecular sciences, 20(4), 916.