Part:BBa_K3037007
Horseradish Peroxidase isoform C (HRP)
| Horseradish Peroxidase | |
|---|---|
| Function | Expression, Reporter |
| Use in | Escherichia coli |
| RFC standard | Freiburg RFC 25 standard |
| Backbone | pSB1C3 |
| Experimental Backbone | pOCC97 |
| Submitted by | Team: TU_Dresden 2019 |
Overview
This BioBrick was made by TU Dresden 2019 team and it is an adaptation of (BBa_K1800002) to the RFC 25 standard in order to make fusion proteins. In the novel fusion protein (BBa_K3037003) this BioBrick was used as a reporter (more information).
HRP was inserted into the plasmid pOCC97 (BBa_K3037000) as vector for expression and characterization in Escherichia coli.
The weight of the protein was calculated based on the base pairs. 924 bp/3 = 308 amino acids, each amino acid weights as average 110 Dalton [1], so the weight of the protein is around 33.88 kDa.
Biology
The metalloenzyme horse radish peroxide is widely used in many biochemical and immunological applications. This Enzyme on its own does not give any visual read out but however upon addition of suitable substrate, HRP oxidizes this substrate and yields a colour change and this can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) that HRP oxidizes. Added advantage of using HRP includes its stability and small size, hence reducing the interference during conjugation reactions such as for secondary antibody detection and moreover it is economical in comparison with other alternative enzymes like the alkaline phosphatase.
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 level 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
Outline
We performed the following characterization experiments:
1) Growth curve of expression with pOOC97 (BBa_K3037000) in E. coli pRARE T7
2) Determination of the total protein amount of cleared lysate after expression assay of the substrate conversion (TMB) compared to (BBa_K1800002)
3) Protein Expression monitoring in SDS-PAGE
4) Activity assay of the substrate conversion (TMB) compared to BBa_K1800002
Experiments in Detail
1) Growth curve of expression with pOOC97 (BBa_K3037000) in E. coli pRARE T7
The HRP was expressed using the plasmid pOOC97 as a backbone (BBa_K3037000)
The purpose of this experiment is to show that the Escherichia coli pRARE T7 grows normaly after the induction of the expression of HRP.
For this the development of the culture was monitored by measuring the OD at 600 nm during different time points before and after induction with 1 mM IPTG. As shown in the curve the growing of the bacteria is not affected by the expression of the protein. Escherichia coli show a the normal growth behaviour as expected in a batch culture.
==== 2) Determination of the total protein amount of cleared lysate after expression assay of the substrate conversion (TMB) compared to (BBa_K1800002) ====
A culture of 100mL Escherichia coli pRARE T7 transformed with the HRP BioBrick inside pOOC97 (BBa_K3037000), they were cultivated until the OD reach 0.5, then the culture was induced with 0.5 mM IPTG and 6 hours after that it was spin down. The pellet was frezeed at -80 degrees and leaved overnight. The next day the cells were lysate and the supernatand was taken for measure the protein concentration. The standard curve was done with the Pierce BCA protein assay kit of thermo scientific (#23225).
Calibration curve (Protein Amount Assay with known BSA concentration)
Values of Total Protein Amounts determined with calibration curve (Values corresponding to the curve shown left)
Total Amounts (Total protein amounts obtained by expression)
Values of Calibration curve (Values corresponding to the curve shown left)
3) Protein Expression monitoring in SDS-PAGE:
A culture of 100 mL Escherichia coli pRARE T7 transformed with the HRP BioBrick inside pOOC97 (BBa_K3037000), they were cultivated until the OD600 reach 0.5. Samples before induction were taken. Then the culuture was induced with 0.5 mM IPTG, samples were taken each 30 minutes (5 samples), then 3 more each 1 hour. Before making the SDS-PAGE, the samples were adjusted to OD of 0.5 to have the same amount of cells in each lane. This way the increase in one specific protein can be observed. The results show the increase of the concentration of the protein in time.
As can be seen in the SDS-PAGE the protein of interest is incresing over time from the point of induction onwards. HRP is marked with a black arrow pointing left.
4) Activity assay of the substrate conversion (TMB) compared to BBa_K1800002
The conversion of transparent TMB substrate to blue reaction product was monitored at 370 nm over half an hour with absorption measurement every 60 seconds.
As can be seen in the graph, the activity of this BioBrick and the original one that it was adapted from are not different. This was to be expected, since the sequences are the same and only the prefix and suffix were changed from RCF10 to RCF25.
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 151
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 393
Illegal XhoI site found at 477 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The Primers used to adapt it to the RFC 25 standard were:
Prefix: GAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAAATGgccggc
Suffix: accggttaaTACTAGTAGCGGCCGCTGCAG
References
[2] Veitch, N. C. (2004). Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry, 65(3), 249-259.
[3] 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.
[4] Humer, D., & Spadiut, O. (2019). Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis. International journal of molecular sciences, 20(4), 916.| None |