Part:BBa_K3589106
Wildtype laccase from uncultured marine bacteria for Escherichia coli
This basic part contains the coding sequence of the wildtype form of a laccase from an uncultured marine bacteria (hereafter referred to as marLac). This part is codon-optimized for Escherichia coli. Combined a promoter and a terminator, this basic part should mediate the oxidation of a wide variety of substrates including phenolic compounds and aromatic amines. This part can be cloned into standard E coli. expression vectors like the commercially available pGEX-6P-1 vector.
This laccase is cold-adapted, thermostable and shows a high tolerance for organic solvents as well as salt (Yang et. al, 2018).
Summary of the Results from Team Kaiserslautern 2020 for part BBa_K3589106
- Expression could be achieved in different E. coli strains when cloned in the pGEX-6P-1 expression vector
- No activity was detected in ABTS-assay
Design of the constructs
For the recombinant expression of the laccase gene from an uncultured marine bacterium (marLac) the E. coli vector pGEX-6P-1 was used (Fig. 1). This expression vector is used to construct a translation fusion protein of Glutathione S-transferase (GST) and our laccase. The expression is regulated by a tac promotor. This promotor combines the strong expression rate from the tryptophan promotor and can be induced with IPTG like the lac operon. To make sure the promotor is inhibited if there is no induction with IPTG the vector also includes the genetic code for the lac-inhibitor that can bind the lac-operon. Because GST has a high affinity for glutathione, the fusion of the laccases with GST allows the purification by affinity chromatography using glutathione agarose. In addition, a protease cleavage site is incorporated between the GST and our fusion protein (MarLac-GST). This allows the separation of GST and the laccase using PreScission Protease. MarLac has a size of 75.8 kDa with and 48.2 kDa without GST. For selection of plasmid containing cells the expression vector carries an ampicillin resistance gene.
In order to be able to do further enzyme assays with the laccase the vector pGEX-6P-1-marlac have to be transformed in the E. coli expression strain BL21(DE3). Additionally, we transformed them in E. coli DH5α for isolation the vectors. The strain has an endA1 mutation. This leads to the inactivity of an intracellular endonuclease, which degrades plasmid DNA. Therefore, plasmid DNA isolation is more efficient.
Fig. 1: pGEX-6P-1_marLac . It includes a tac promotor that can be regulated with the lac-inhibitor (expressed with lacI) which can be inactivated with IPTG. The protein is fused to GST, with a PreScission protease cleavage site in between. The vector has an ampicillin resistance gene, which is used for selection.
Growth test
First, we wanted to find out the best conditions for the plasmid containing E. coli BL21(DE3) cells to produce marLac. We did a test expression where the cells grow at different temperatures (37°C, 30 °C and 17°C; Fig. 2). After every hour we took a sample for a SDS-PAGE (Fig. 3) and measured the optical density (OD) at 600 nm. We designed the experiment based on Kittl et al., 2012 where they used copper sulfate in the media for the protein production. So, we tested the growth at all the temperatures, one time with CuSO4 and one time without, to be sure that the copper sulfate doesn’t influence the growth (Fig. 2).
To see if our protein is soluble or insoluble, we lysed the cells and separated the pellet and the soluble fraction with SDS-PAGE (Fig.3).
Fig. 2: Growth curve from E. coli BL21(DE3) pGEX-6P-1_marLac producing cells at different temperatures. The expression was done over 19 hours, the cells were induced at x=0. The growth of E. coli BL21(DE3) pGEX-6P-1_marLac is shown. It was tested at 37°C, 30°C and 17°C and all temperatures both with [w] and without CuSO4.
Fig. 3: SDS-PAGE of the test expression with different temperatures. Samples of E.coli BL21(DE3) pGEX-6P-1_marLac were taken before induction and after inducing with IPTG after every hour for every temperature. The LB medium contains CuSO4.The cells were disrupted by sonication and insoluble and soluble fraction were separated. The red boxes show the produced marLac. Marker: New England BioLabs ® Blue Protein Standard Broad Range. The proteins sought are at the level of a relative molecular mass of the marker at and 75 kDa (marLac, size 75.8 kDa). The positive control is a GST fusion protein with a size of 26 kDa. .
Furthermore, we wanted to test in which medium the cells grow best. We compared LB-Medium with 2YT-Medium in two different temperatures (37°C, 30°C; Fig. 4)
(B)
Fig. 4: SDS-PAGE and western blot of the test expression with LB medium and 2YT medium. marLac producing cells growing in LB medium is shown in (A) and 2YT medium in (B). Each medium contains CuSO4. After induction, the cells grow at 37°C and 30°C for 3 h and 5 h. The cells were disrupted by sonification and insoluble and soluble fraction were separated. The red boxes show the produced marLac. The fusion protein (marLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). Marker: New England BioLabs ® Blue Protein Standard Broad Range.
As it is shown in Figure 3 and 4 the protein seems to be insoluble. So, we took the conditions where we had the biggest amount of soluble protein. Because of that we decided to produce marLac at 37 °C for 3 h (Fig. 3) in 2YT-Medium (Fig. 4). Because the laccase is an enzyme with copper-centers and the cells growth isn’t inhibited by the copper sulfate (Fig. 2), we decided to use CuSO4 in our production medium every time.
Production and Purification
With the knowledge about the best conditions we started the production of our protein in the transformed E. coli BL21(DE3) strain. The expression was induced with IPTG. After the respective times (marLac 37 °C for 3 h) we harvested the cells. To purify the protein we lysed the cells and worked on with the soluble proteins in the cytoplasma. The first step of purification is to separate the translation fusion protein from other soluble proteins using affinity chromatography (Fig. 5a). After dialysis with PreScission Protease a second affinity chromatography removes the GST from the solution (Fig. 5b). Then our purified protein was ready to be tested for activity. For marLac we got around 0.33 mg protein per gram cell wet weight.
(A)
(B)
Fig. 5: Purification of marLac with affinity chromatography. The cells grew at 37°C and were harvested after 3 h then lysed with ultrasonic and centrifuged to receive the soluble fraction. The marLac in the soluble fraction was purified with glutathione-agarose affinity chromatography. The SDS-PAGE shows the samples taken after every step. The fusion protein (marLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). (A) shows the first step of the purification before dialysis with PreScission Protease. The cell lysate was applied to the column. The fusion protein with GST-tag was able to bind to the column, the remaining lysate passed the column (flow through). This was followed by a washing step with washing buffer. Finally, the fusion protein was eluted with elution buffer containing glutathione (eluates 1-6). This was followed by dialysis with the PreScission protease to separate the laccase from the GST-tag. (B) shows the second step after dialysis. The GST binds to the glutathione agarose due to its affinity. The laccase flows through the column (D1-3). After a washing step with PBS (D4), the GST is eluted using a washing buffer containing glutathione (elution). The produced marLac is shown in the red boxes. Marker: New England BioLabs ® Blue Protein Standard Broad Range.
ABTS-Activity Assay for E. coli marLac:
The marine laccase marLac was an alternative that we were excited to try due to its optimal pH being at 7, closer to the average wastewater pH and more stable.1 In the assays, we noted that there appeared to be no significant activity however, even when adjusting parameters such as growing conditions, salt levels, and temperature.
We noted that when pipetting the samples in, often the solution would be extremely foamy, an indicator of denatured proteins. This would lead to less sample in each well or delays in beginning the assay, which could have been a source of error in our measurements. The samples would also often elute proteins during the course of the assay, causing any increase in the actual measurement to likely be a result of this rather than actual ABTS oxitation occurring.
The final assay done with marLac (23.85 μM obtained in less than 200 μL solution, so a negative ABTS control was not possible, see Fig. 7) was a replication of our initial protocol to verify concentration obtained by Bradford and check if too many factors had been adjusted. The positive control was T. versicolor at our highest tested concentration (3570 μM) due to the poor activity this laccase had at pH 7.
Fig. 6: ABTS assay for both marLac and BaLac (strain AD494). The assay was performed in pH 7 and 4 Phosphate-Citrate buffer respectively to provide the manufactured enzymes optimal conditions. Positive (+) control is both 3570 μM in pH 7 buffer (C1) for marLac and 40 μM T. versicolor in pH 4 buffer (A1) for BaLac. All wells except D1, B3, and B4 contained 250 μM ABTS to act as an ABTS negative control. There was not enough produced enzyme of marLac to run an ABTS negative control. Negative (-) controls contained no enzyme, and tested ABTS against both pH 4 (A4) and pH 7 (B4). A light blue tint in the BaLac well (A3) indicates a reaction over the 4 hour assay.
Fig. 7: marLac ABTS assay analysis. a) Raw data: Positive (+) control is 3570 μM T. versicolor. Negative (-) control contains no enzyme. The marLac sample had its concentration (23.85 μM) determined by Bradford assay and shows similar activity to negative control, beginning at a slightly higher absorbance likely due to eluting proteins. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates the change from initial to visualize the reaction more clearly, showing the marLac sample reacting at similar rate as the negative sample over the course of 4 hours.
No activity was found in any tested marLac sample due to the complications with insoluble proteins and low concentrations, so further investigation is required to implement this construct.
References
(1) Yang, Q.; Zhang, M.; Zhang, M.; Wang, C.; Liu, Y.; Fan, X.; Li, H. Characterization of a Novel, Cold-Adapted, and Thermostable Laccase-Like Enzyme With High Tolerance for Organic Solvents and Salt and Potent Dye Decolorization Ability, Derived From a Marine Metagenomic Library. Front. Microbiol. 2018, 9, 2998. https://doi.org/10.3389/fmicb.2018.02998.
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