Team:Uppsala Universitet/Results


Cloning Results

Gene fragments that include coding regions for horseradish peroxidase (HRP), lignin peroxidase (LiP), glyoxal oxidase (GLOX), manganese peroxidase (MnP), HRP_2A_eGFP, HRP_2A_AAO and AAO_2A_eGFP were assembled to shuttle vector (pPICZαB) using Gibson assembly.

Colony PCR on transformed Escherichia Coli was used to amplify inserted gene fragments for gel electroporation analysis. Seven constructs: pPICZαB - LiP, HRP_2A_eGFP, GLOX, MnP, HRP, HRP_2A_AAO & AAO_2A_eGFP were shown to have the right band size, as shown in Figure 1 and they were all sequence verified to be the correct sequences.

Figure 1: Gel verification of gene fragments amplified by Colony PCR.
LiP: expected band size 1644 bp; HRP_2A_eGFP: expected band size 2367 bp; GLOX: expected band size 2205 bp; MnP: expected band size 1662 bp; HRP: expected band size 1590 bp; AAO_2A_eGFP: expected band size of 3087 bp; HRP_2A_AAO: expected band size 3699 bp.

Protein Expression

In order to determine the correct laboratory conditions for the Pichia pastoris expression system, the pPICZαB_RV1284 plasmid was used. Previous experiments showed high expression and secretion of the RV1284 protein product of this plasmid.

P. pastoris cells were grown and transformed with the plasmid via electroporation. Expression cultures were inoculated from the transformants and grown for approximately 7 days and induced with 0.5 % methanol every 24 h. Cell and supernatant fractions were analysed by 15 % SDS-PAGE (Figure 2). After induction, a prominent band at the calculated size of RV1284 is visible in the supernatant but absent in the uninduced supernatant. Therefore, it was concluded that RV1284 was successfully expressed and secreted, confirming that the established conditions work. Thus, experimental focus was moved to expression of functional enzymes for lignin degradation.

Figure 2: Expression and secretion of RV1284 in KM71H Culture V
KM71H P. pastoris cells were transformed with pPICZαB_RV1284, induced (i), fractionated into pellets (P) and supernatants (S), and analysed by 15 % SDS-PAGE stained with Coomassie Blue. u, uninduced In the induced supernatants, a band is visible at around 17 kDa showing secretion of RV1284.

Figure 3: Expression and secretion of Manganese Peroxidase (MnP)
X-33 P. pastoris cells were transformed with pPICZαB_MnP, induced (i), fractionated into pellets (P) and supernatants (S), and analysed by 10 % SDS-PAGE stained with Coomassie Blue. u, uninduced Induction bands are visible at approximately 70 kDa for both P and S (red arrows), showing secretion of MnP.

Successful Expression of Manganese Peroxidase in P. pastoris

Five basic constructs were created by Gibson assembly; pPICZαB_HRP, pPICZαB_AAO, pPICZαB_MnP, pPICZαB_Glox and pPICZαB_LiP. P. pastoris cells were again transformed via electroporation and expression cultures were inoculated. Fractions were analysed by 10 % SDS-PAGE. Culture III of X-33 with pPICZαB-MnP showed an induction band in cell pellets and supernatants, indicating the successful expression and secretion of Manganese peroxidase (MnP) (Figure 3). However, other expression cultures lacked an induction band (not shown), indicating the procedure needed to be optimized. We proceeded to optimize the expression by adding a 2A peptide.

Generation of easy-screen coexpressing P. pastoris cell lines

To generate easier screens for protein expressions, constructs with the 2A self-cleaving peptide and eGFP reporter gene were utilised, read more about this in the design page. The transformed P. pastoris KM71H cells were then restreaked on a methanol-containing agar plate to induce expression. eGFP was visualised by excitation on a blue light box under an orange filter (Figure 4). In total, 50 colonies were screened by this method. Only 4 of 50 colonies were eGFP positive, showing the importance of this screening technique to obtain the clones that successfully expressed the gene of interest.

Figure 4: eGFP coexpression from 2A-constructs
Transformed P. pastoris KM71H cells coexpressing eGFP and AAO were screened on a BMMY agar plate. eGFP is visible for two of the restreaked colonies, in the middle. Picture was taken on a blue light box under an orange filter after 4 days of incubation at 28 °C with daily addition of 50 µL methanol on the plate lid.

Figure 5: Expression of Horseradish Peroxidase (HRP) and Aryl-alcohol oxidase (AAO)
X-33 P. pastoris cells were transformed with pPICZαB_HRP-2A-AAO and expression cultures were induced. Different fractions (pellet (P) and supernatant (S) samples / uninduced (u) and induced (i) cultures) from X-33 HRP-2A-AAO expression culture were analysed on a 10 % SDS-PAGE stained with Coomassie Blue. After 24 h an induction band can be seen at around 55 kDa, which is approximately the calculated molecular weight for both HRP and AAO. This shows that the enzymes are expressed as well as that the cleavage initiated by the 2A-peptide is functional

Coexpression of Horseradish peroxidase and Aryl-alcohol oxidase

In addition to utilising the 2A self-cleaving peptide for screening methods, it was also used to coexpress two enzymes, HRP and AAO. The transformed P. pastoris cells had to be screened by SDS-PAGE (Figure 5). A very prominent induction band is visible in the pellet, at approximately 55 kDa corresponding to the calculated molecular weights of both HRP and AAO. This shows that one or both of the enzymes were expressed and that the cleavage initiated by the 2A-peptide was functional.

Proof of enzymatic activity

Enzymatic activities of pellet lysate from X-33 pPICZαB_HRP-2A-AAO was measured using different enzymatic assays for HRP and AAO (Figure 6A and B, respectively). The HRP assay showed a rapid increase in the induced cell lysate, while the uninduced cell lysate displayed no activity. For both enzymes, the difference in activity between uninduced and induced samples was significant. Thus, the induction band in Figure 5 contains both enzymes.

Figure 6: Assays of cell lysate for HRP and AAO enzymatic activities
The activities of the two enzymes expressed in Figure 5 were assessed using two different enzymatic assays. Uninduced, orange; induced, green. Assays were performed in triplicates. (A) ABTS-assay for HRP. (B) FOX-assay for AAO activity. Two different volumes of lysate were used and absorbance at 560 nm was measured. The difference in activity between uninduced and induced samples was statistically significant as measured by paired t-test (*= p<0.05 and ****= p<0.00001, respectively).

The obtained results show that in total 5 different proteins were expressed successfully (RV1284, MnP, HRP, AAO and eGFP). Also, a method for efficient screening of protein-expressing P. pastoris clones was established, while also verifying the functionality of the 2A self-cleaving peptide in P. pastoris.


Proof Of Concept

Enzymatic Assay

Before expressing the enzymes in yeast, the assays were tested on enzymes acquired in other ways.

HRP extracted from horseradish

Due to bottlenecks in enzyme supply, HRP was isolated from locally purchased horseradish. Crude extract was prepared by blending, sonication and subsequent ammonium sulphate precipitation (30 % and 65 % saturation). 65 % ammonium sulfate fraction was resuspended in 50 mM phosphate buffer pH 7,5. Enzyme activity was confirmed by ABTS-assay, which led to an immediate change in the color (Figure 7A).The HRP oxidizes the ABTS which leads to a colorimetric change of the sample. When the assay was tested on the extracted HRP, a dilution of the sample was made to 0.0005 before applied to the cuvette and tested with spectrophotometer (Figure 7B). The HRP oxidizes the ABTS which leads to a colorimetric change of the sample.

Figure 7: Activity of horseradish peroxidase extracted from horseradish.
(A) Color-change after addition of undiluted crude Horseradish extract into ABTS reaction mixture. (B) The increase in absorbance indicates HRP activity.

Figure 8: ABTS-assay of bought HRP
The figure shows ABTS-based assay on commercial HRP with absorbance plotted (405 nm) against time (s). A clear increase in absorbance after addition of enzyme indicates that activity is detectable.

Commercial HRP

After commercial HRP was obtained, an ABTS-based assay was performed to assess the activity (Figure 8). A dilution of 1:200 for the bought HRP was made before applying the sample and working reagent into a cuvette and analyzed in a spectrophotometer. The result indicates that the assay is able to detect the presence of HRP, which in turn means that the activity of yeast-expressed HRP would be detectable.

Magnese Peroxide enzymatic assay

Two different enzymatic assays were tested to detect manganese peroxide activity. Commercial MnP was used as a positive control, however, activity could not be shown. This indicates that either the enzyme is not active or the assays did not work.

Lignin Degradation and Detection

Lignin degradation was carried out by both mechanical force (sonication) and enzymatic reactions (HRP). Sonication was used to see if it resulted in any detectable changes to the lignin sample. The detection of lignin degradation was performed with: Native-PAGE, SDS-PAGE, TLC-plate and UV-Vis analysis. The lignin displayed in the result section is Kraft Lignin, an industrial kind of lignin which was acquired from Stora Enso.

Full spectrum UV-Vis spectroscopic analysis

If full spectrum analysis could show a difference between sonicated lignin and non-sonicated lignin, the method could potentially be suitable for detection of enzymatic degradation of lignin. The full spectrum analysis showed a clear distinction between sonicated lignin and non-sonicated lignin (Figure 9). The absorption spectrum of the sample had been altered, and the absorption maximum in the sample changed from 230 nm to 365 nm after sonication treatment. These results indicate that changes in the sample are detectable by full spectrum UV-Vis spectroscopic analysis.

Figure 9: Full spectrum analysis of sonicated and non-sonicated Kraft Lignin.
The analysis shows a difference in absorption spectrum between the sonicated lignin and the non-sonicated lignin. The absorption maximum in the lignin sample changed from 230 nm to 365 nm after sonication.

Figure 10: Native-PAGE analysis of fractionated lignin.
Different lignin samples acquired through fractionation run on a Native-PAGE gel. The gel did not show a difference in migration speed between the samples.

Native-PAGE analysis of Kraft Lignin fractionation

The fractionation was performed to separate the heterogeneous lignin sample into more defined fractions in terms of size. This would have made size exclusion chromatography a more viable method for detection of lignin degradation. The fractions were run on a Native-PAGE to see if any size difference could be detected with this method (Figure 10). The gel did not give results that indicated changes to size.

SDS-PAGE analysis of enzymatically treated Kraft Lignin

After the samples were treated with HRP (commercial), the goal was to detect degradation of lignin. SDS-PAGE was utilized for this purpose, and the gel indicated no difference between lignin samples treated with HRP for different lengths of time (Figure 11). The SDS-PAGE was visualized with UV-light and a ceramic TLC-plate under the gel which made the lignin traces visible. When the gel is observed on the ceramic TLC-plate and UV-light is shined upon it, the smears from the lignin samples can be seen in the gel when the TLC-plate fluoresces green. No visible change was observed when sonicated lignin was applied to the gel.

Figure 11: SDS-PAGE gel of HRP-treated Kraft Lignin.
Lignin sample was treated with active HRP (commercial) and incubated for different amounts of time with the active enzyme. The lignin was visible on the gel, however, no significant difference could be observed between the samples.

Figure 12: Different kinds of lignin and monolignols on TLC-plate.
The different kinds of lignin loaded together with monolignols were loaded on a TLC-plate. The samples showed different migration patterns on the TLC-plate.

TLC plate analysis of enzymatically treated Kraft Lignin

Since lignin degradation could lead to formation of structures with different properties, TLC-analysis was used to see if changes to the solubility or polarity of the lignin had been affected. Different kinds of lignin together with monolignols were run on a TLC-plate (Figure 12). The aim was to observe if a difference could be observed between the samples. Since anisyl alcohol and coniferyl alcohol are compounds which form the lignin structure, an experiment that differentiates them from lignin could potentially be used to detect lignin degradation. The experiment indicated that different compounds that make up lignin are distinguishable on thin layer chromatography.

Treatment of Kraft Lignin with HRP

With the goal to enzymatically degrade lignin, bought HRP and hydrogen peroxide was added to lignin in water. A change in color was observed in the samples over time, which could be indicative of a change in the lignin structure (Figure 13). The reaction was stopped and the samples stored. After 60 days, the untreated lignin still shows a lighter color than observed in the HRP-treated lignin samples (Figure 14).

Figure 13: Three samples of Kraft Lignin treated with bought HRP.
The samples have been exposed to HRP for different amounts of time. A change in color can be observed in the samples that have been exposed to HRP for a longer time.
Figure 14: Three samples of Kraft Lignin treated with bought HRP.
The samples of lignin with the enzymes are the same as Figure 13, but the samples were observed after 60 days. The HRP-treated lignin still shows a darker color than the untreated samples.