Part:BBa_K3037007

Horseradish Peroxidase isoform C (HRP)

Horseradish Peroxidase
Function	Expression
Use in	Escherichia coli
RFC standard	RFC 25 compatible
Backbone	pSB1C3
Submitted by	Team:TU_Dresden 2019[1]

Overview

The TU Dresden 2019 team design this biobrick in order to make the fusion protein BBa_K3037003 in accordance to the RFC 25 standard. (more information) In this protein HRP was used as a reporter.

HRP was inserted into the pSB1C3 vector for transformation and expressed in Escherichia coli.

https://parts.igem.org/Assembly_standard_25

This Biobricks was made from BBa_K1800002 but adapted to the RFC 25 standard

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)

Characterization

[2]

Sequence

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 151
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 393
  Illegal XhoI site found at 477
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Refenrences

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

[2] 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.

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