Difference between revisions of "Part:BBa K3872001"
 
Line 16: Line 16:
 
]]
 
]]
  
−
We transformed <i>E. coli</i> BL21(DE3) cells with pET36b-<i>cueO</i> expression constructs. Three colonies from that strain were analyzed by restriction digestion with NdeI and XhoI. Two of them were true transformants, as seen in the Figure 1. The protein production of the cells was induced by IPTG, the cells were lysed by sonication and His-tagged laccases were extracted with nickel affinity chromatography. The sizes of purified proteins were verified with SDS-PAGE, which showed clear bands around 60 kDa, expectedly corresponding to the calculated size of CueO (Fig. 2). That suggests that CueO was successfully produced by generated <i>E. coli</i> BL21(DE3) strains.
+
We transformed <i>E. coli</i> BL21(DE3) cells with pET36b-<i>cueO</i> expression constructs. Three colonies from that strain were analyzed by restriction digestion with NdeI and XhoI. Two of them were true transformants, as seen in the Figure 1. The protein production of the cells was induced by IPTG, the cells were lysed by sonication and His-tagged laccases were extracted with nickel affinity chromatography. The sizes of purified proteins were verified with SDS-PAGE, which showed clear bands around 60 kDa, expectedly corresponding to the calculated size of CueO (Fig. 2). That suggests that CueO was successfully produced by generated <i>E. coli</i> BL21(DE3) strain.
  
 
[[Image:T--Aboa--fig3.jpg|thumb|400px|<b>Figure 2. SDS-PAGE of raw extracts and purified enzyme fractions.</b> Bio-Rad Ready Gel® Precast Polyacrylamide Gel was used and the ladder was the PageRuler Unstained Protein Ladder (Thermo Fisher).
 
[[Image:T--Aboa--fig3.jpg|thumb|400px|<b>Figure 2. SDS-PAGE of raw extracts and purified enzyme fractions.</b> Bio-Rad Ready Gel® Precast Polyacrylamide Gel was used and the ladder was the PageRuler Unstained Protein Ladder (Thermo Fisher).

Latest revision as of 15:08, 21 October 2021


Multi-copper oxidase CueO from Escherichia coli

The part codes for a laccase from Escherichia coli. It is an enzyme that catalyses oxidation reactions of many types of compounds, e.g. different pharmaceuticals and dyes.

Background

Laccases are multi-copper oxidases (MCOs) that can catalyze reactions of many substrates, for example organic compounds, dyes and pharmaceuticals. They can be found in fungi, bacteria, insects and plants and they are involved in many different functions, for example in lignin degradation, pigmentation, pathogenesis of fungi and wound healing in plants. [1] CueO is a multicopper oxidase laccase produced by E. coli [2].

In vitro, CueO can oxidise various compounds, such as catechols, ferrous iron and iron-chelating siderophores [3]. It has been proven that CueO plays a pivotal role in regulating the copper resistance of E. coli [4]. The mechanism of action of CueO in vivo has been suggested to be the cuprous oxidation in which CueO confers more toxic Cu(I) to less toxic Cu(II). According to one study, the optimal pH for the proper CueO function is 6,5. [2]

Protein production and purification

Figure 1. The verification digestions of the pET36b-cotA, pET36b-cueO and pET36b-yak expression constructs. The expression constructs were digested with NdeI and XhoI restriction enzymes. The 1 kb DNA ladder (NEB) was used as a molecular weight standard, the DNA was colored with SyBR Safe DNA Gel Stain (ThermoFisher), and the gel was visualized under UV light. The agarose gel used was 1.2 %.

We transformed E. coli BL21(DE3) cells with pET36b-cueO expression constructs. Three colonies from that strain were analyzed by restriction digestion with NdeI and XhoI. Two of them were true transformants, as seen in the Figure 1. The protein production of the cells was induced by IPTG, the cells were lysed by sonication and His-tagged laccases were extracted with nickel affinity chromatography. The sizes of purified proteins were verified with SDS-PAGE, which showed clear bands around 60 kDa, expectedly corresponding to the calculated size of CueO (Fig. 2). That suggests that CueO was successfully produced by generated E. coli BL21(DE3) strain.

Figure 2. SDS-PAGE of raw extracts and purified enzyme fractions. Bio-Rad Ready Gel® Precast Polyacrylamide Gel was used and the ladder was the PageRuler Unstained Protein Ladder (Thermo Fisher).

Characterization and results

To measure the laccase activity, we performed a kinetic laccase assay using syringaldazine as substrate. The reaction is based on substrate oxidation which can be spectrophotometrically monitored as signal increase at 530 nm. However, the CueO elution fraction didn't seem to be active towards syringaldazine.
















Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[1] L. Arregui et al. Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories 2009, 18 (1) 200, https://doi.org/10.1186/s12934-019-1248-0.
[2] S. Roberts et al. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (5) 2766-2771, https://doi.org/10.1073/pnas.052710499.
[3] S. Singh et al. Cuprous Oxidase Activity of CueO from Escherichia coli. Journal of Bacteriology 2004, 186 (22) 7815-7817, https://doi.org/10.1128/JB.186.22.7815-7817.2004
[4] G. Grass and C. Rensing. CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochemical and biophysical research communications 2001, 286 (5) 902-908, https://doi.org/10.1006/bbrc.2001.5474.