Difference between revisions of "Part:BBa K3037007"

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 a suitable substrate, HRP oxidizes this substrate and yields a colour change that can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) that HRP oxidizes. Additionally, an advantage of using HRP includes its stability and small size, hence reducing the interference during conjugation reactions such as for secondary antibody detection. Moreover, it is very economical in comparison with other alternative enzymes like Alkaline Phosphatase.

HRP is an extensively studied and one of the most important enzymes obtained from plants. One of the main reasons why this enzyme is of great interest 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) commonly using 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 to produce color changes in specific substrates. Therefore, HRP has many industrial applications, especially in biosensors and diagnostic kits (e.g., immunoassays, ELISA, EMSA…). (Krainer, F. W. et al 2014)

Furthermore, HRP has many characteristics that makes 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 being used to improve the properties of HRP. (Veitch, N. C. 2004). The commercially available HRP is extracted from Armoracia rusticana roots. However, Armoracia rustica requires long cultivation time 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 such 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 in pOCC97 (BBa_K3037000) in E. coli pRARE T7

2) Determination of the total protein concentration of cleared lysate after expression assay of the substrate conversion (TMB) compared to (BBa_K1800002)

3) Protein Expression monitored via SDS-PAGE

4) Activity assay of the substrate conversion TMB compared to (BBa_K1800002)

Experiments in Detail

1) Growth curve of expression in pOCC97 (BBa_K3037000) in E. coli pRARE T7

The HRP was expressed using the plasmid pOCC97 as a backbone (BBa_K3037000)

The purpose of this experiment was to show that the Escherichia coli pRARE T7 grows normally after the induction of HRP expression.

For this, growth of bacteria was monitored by measuring the Optical absorbance at 600 nm during different time points before and after induction with 1 mM IPTG. As shown in the following Figure, the Escherichia coli growth is not affected by the expression of the protein. It shows a normal growth behaviour as expected in a batch culture (Figure 1).

Figure 1: Growth curve of Escherichia coli before and after expressing HRP. Vector pOCC97 (BBa_K3037000). Induction with 1mM IPTG

2) Determination of the total protein concentration of cleared lysate after expression assay of the substrate conversion (TMB) compared to (BBa_K1800002)

In order to determine the total protein content of the cleared lysate after the expression the following assay was performed. First, the standard curve was done with the Pierce BCA protein assay kit of Thermo Scientific (#23225) (Figure 2).

Figure 2: Calibration curve with known BSA concentrations in order to determine the concentrations of our samples.

Then different cultures of E. coli pRARE T7 transformed with different BioBricks using pOCC97 (BBa_K3037000) as a vector were set. The BioBricks used were:

• A fusion protein of MBP and HRP (BBa_K3037008)
• This HRP but in the RFC10 standard (BBa_K1800002)
• This HRP adapted to the RFC25 standard (BBa_K3037007)

Then a culture of 100 mL E. coli pRARE T7 transformed with the different BioBricks was cultivated until the OD reached 0.5, then the culture was induced with 0.5 mM IPTG and 6 hours after that it was spun down. The pellet was stored at -80 degrees and left overnight. The next day the cells were lysed and the supernatant was taken to measure the protein concentration. The results were compared with the standard curve to calculate the concentration as shown in Figure 3.

Figure 3: Protein concentration of HRP measured in the cleared lysate of E. coli pRARE T7 carrying the expression backbone pOCC97 (BBa_K3037000) with different inserts. Expression was induced 0.5 mM IPTG

3) Protein Expression monitoring in SDS-PAGE:

A culture of 100 mL Escherichia coli pRARE T7 were transformed with the HRP BioBrick inside pOCC97 (BBa_K3037000), they were cultivated until the absorbance reached 0.5. Samples before induction were taken. Then the culture was induced with 0.5 mM IPTG,every 30 minutes samples were collected (5 samples), then 3 more each 1 hour. Before making the SDS-PAGE, the samples were adjusted to absorbance of 0.5 to have the same amount of cells in each lane. By doing so, the increase of a specific protein can be observed. The results show the increase of the concentration of the protein in time (Figure 4).

Figure 4: SDS-PAGE of the expression of HRP (BBa_K3037007) in the vector pOCC97 (BBa_K3037000). Upper anotations showing the different times of induction in minutes. The protein band is marked with an arrow

As it can be seen in the SDS-PAGE, the protein of interest is increasing 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 measurements every 60 seconds for the HRP adapted to the RFC10 standard (BBa_K1800002) and to the RFC25 (BBa_K3037007) (Figure 5)

Figure 5: Activity assay of HRP in different iGEM standards expressed in pOCC97 (BBa_K3037000) monitored at 370 nm measuring every 60 seconds.

As it can be seen in the graph, the activity of this BioBrick and the original one from which it was adapted from are not different. This was our expected result, since the sequences are the same and only the prefix and suffix were changed from RCF10 to RCF25.

Sequence

Design Notes

The Primers used to adapt it to the RFC 25 standard were:

Prefix: GAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAAATGgccggc

Suffix: accggttaaTACTAGTAGCGGCCGCTGCAG

References

[1] https://www.promega.com/~/media/files/resources/technical%20references/amino%20acid%20abbreviations%20and%20molecular%20weights.pdf

[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.