Welcome to the Engineering page, where we present the progress and results of our project based on the concepts of synthetic biology. Here, we focus on the conceptual development and engineering cycle of the entire project and present our progress in the order of "Brief Introduction", "Design", "Build", "Test ", and "Learn" to present the progress of our project.
Certain fusion tags are highly soluble and can facilitate soluble expression of proteins by binding and isolating aggregation-prone folding intermediates of their target proteins[1].
We chose the following two fusion tags to incorporate into the original target protein to address the inclusion body issue.
Thioredoxin A (TrxA): thermally stable, highly soluble and with solid folding properties.
N-utilization substance A (NusA): a highly soluble protein that binds and segregates the aggregation-prone folding intermediates of its target proteins, preventing them from self-association and aggregation.
We insert both the target protein and the DNA of the fusion tag into the plasmid, which is then transcribed and translated to produce the target protein attached to the fusion tag.
We synthesized the pET-22b(+)-LSPETase plasmid from the company and will construct the recombinant plasmid by attaching each of the above two fusion tags in front of the LSPETase.
Plasmid construct design: (Gibson assembly)
TrxA and NusA gene sequences were inserted into the vector pET-22b(+)-LSPETase, and the constructed recombinant plasmids were named pET-22b-TrxA-LSPETase; pET-22b-NusA-LSPETase.
Pairs of strains modified with were named TrxA-LSPETase; NusA-LSPETase.
The vector pET-22b(+)-LSPETase was first linearised by inverse PCR, and the fusion-tagged fragment was amplified by post-PCR, and the plasmid was mapped as follows:
Fig. 1 Plasmid mapping of pET-22b-TrxA-LSPETase
Fig. 2 Plasmid mapping of pET-22b-NusA-LSPETase
Section 1: The DNA fragment of the fusion tag is amplified by PCR and the vector is linearized by reverse PCR.
Section 2: After homologous recombination of the linearised vector and amplified fragments, heat-stimulated transformation is performed.
Section 3: Single colonies of bacteria grown on the transformed plates were picked and cultured, then colony PCR was performed to verify that the single colonies grown contained the recombinant plasmid.
Section 4: Bacteria with successful colony PCR validation were sent for testing, and bacteria with correct sequencing were cultured for induced expression. After induction of expression, the supernatant and precipitate after cell crushing were subjected to SDS-polyacrylamide gel electrophoresis, respectively.
Fig. 3 SDS-PAGE of recombined LSPETase with fusion tag attached (M: Marker; Lane 1: Supernatant of NusA-LSPETase slurry; Lane 2: Precipitate of NusA-LSPETase slurry; Lane 3: Supernatant of TrxA-LSPETase slurry; Lane 4: Precipitate of TrxA-LSPETase slurry; lane 5: Supernatant of original LSPETase slurry; lane 6: Precipitate of original LSPETase slurry.)
The molecular weight of LSPETase is 30.2 kDa, while NusA-LSPETase and TrxA-LSPETase are 87.0 kDa and 43.8 kDa, respectively.
As depicted in Fig. 3, original LSPETase (lanes 5 and 6) had less supernatant and more precipitation in the cell breakage solution, indicating that the soluble expression of the target protein was restricted. After the addition of fusion tag NusA (lanes 1 and 2), the concentration of the 87.0 kDa protein band (lane 1) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band (lane 2) was obviously lightened. And after the addition of fusion tag TrxA (lanes 3 and 4), the concentration of the 43.8 kDa protein band (lane 3) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band was obviously lightened.
Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analysed as follows:
Fig. 4 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the fusion tag
Fig. 5 Percentage comparison of the optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the fusion tag
An examination of Figure 4 and Figure 5 revealed that, under the specified induction conditions, the protein solubility of TrxA-LSPETase and NusA-LSPETase, exhibited a remarkable enhancement, with a respective increase of 3.1-fold and 1.7-fold compared to LSPETase. Moreover, the density values of the protein bands in the precipitate exhibited a reduction. This provides additional validation for the inference that the incorporation of fusion tags effectively facilitates the solubility of the target protein.
The utilization of a fusion tag positioned at the N-terminus of LSPETase aimed to enhance the solubility of the protein within E. coli. However, we seek to explore alternative approaches that may yield greater effectiveness. Through an extensive review of relevant literature, we have discovered that the intracellular reducing environment hampers the formation of protein disulfide bonds, resulting in the aggregation and formation of inclusion bodies in proteins expressed at high levels. Conversely, the oxidative environment present in the periplasmic space of E. coli promotes the formation and stable existence of disulfide bonds. Therefore, directing the LSPETase enzyme to the periplasmic space emerges as a promising strategy to achieve soluble expression.
Particular signal peptides possess the ability to convey synthesized proteins to the periplasmic compartment. The sequestration of proteins expressed in the periplasmic space offers advantages such as protein purification and prevention of interactions with cytoplasmic constituents.
Consequently, by employing a rational design approach and incorporating signal peptide sequences, our objective is to establish a system for co-expressing signal peptides and LSPETase enzymes, enabling the targeted transport of recombinant proteins to the periplasmic domain for secretory expression. This approach holds the potential to address the challenge of inclusion body formation during the expression of exogenous proteins.
Disulfide Bond Formation Protein A (DsbA) and Outer Membrane Protein A (OmpA) represent two N-terminal signal peptides of approximately 20-30 amino acids. Both DsbA and OmpA possess the capability to facilitate the transfer of synthesized proteins to the periplasmic intermediates. Notably, DsbA exhibits an additional role in catalyzing the formation of disulfide bonds within newly synthesized proteins[2], thereby promoting their soluble expression. Hence, DsbA serves as a catalyst for the formation of disulfide bonds, enabling the soluble expression of nascent proteins.
In the initial design, since the signal peptide is a small molecule protein of 20-30 amino acids, we aimed to design primers to add the coding sequence of the signal peptide to the front of the coding sequence of the LSPETase enzyme by PCR. To achieve this, we employed a reverse PCR primer, positioned between the RBS and LSPETase enzyme sequences to append the signal peptide sequence to the primer.
Subsequently, as we constructed the plasmid through homologous recombination, we linearized the plasmid through reverse PCR and added signal peptide sequences during this process, ensuring that the two ends of the linearized plasmid fragment had approximately 20 bp homologous sequence regions, enabling it to achieve single fragment homologous recombination.
We synthesised the pET-22b(+)-LSPETase plasmid and constructed the recombinant plasmid by attaching each of the above two signal peptides in front of LSPETase.
Plasmid construct design: (Gibson assembly)
DsbA and OmpA gene sequences were inserted into the vector pET-22b(+)-LSPETase, and the constructed recombinant plasmids were named pET-22b-DsbA-LSPETase; pET-22b-OmpA-LSPETase.
Pairs of strains modified with were named DsbA-LSPETase; OmpA-LSPETase.
By using reverse PCR to linearize the vector pET-22b (+) - LSPETase and attach signal peptide fragments, a single fragment homologous recombination plasmid was constructed. The designed plasmid map is as follows:
Fig. 6 Plasmid mapping of pET-22b-DsbA-LSPETase
Fig. 7 Plasmid mapping of pET-22b- OmpA -LSPETase
The steps are similar to adding a fusion label at the N-terminal of LSPETase: the vector is linearized through reverse PCR, but at the same time, the linearized plasmid must be accompanied by a signal peptide sequence. The linearized plasmid with the signal peptide must undergo homologous recombination of a single fragment and undergo heat shock transformation. Cultivate the transformed bacteria to induce expression. The expressed protein was subjected to SDS-polyacrylamide gel electrophoresis to analyze whether the bands were correct and verify the effectiveness of the experiment.
Fig. 8 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h (M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of DsbA-LSPETase fermentation broth; lane 4: Supernatant of OmpA-LSPETase fermentation broth)
As depicted in Figure 8, it was observed that upon induction of expression, the LSPETase enzyme, when equipped with the N-terminal signal peptide, did not exhibit localization within the fermentation broth. Upon revisiting the relevant literature, it was hypothesized that the recombinant target protein was likely directed to the periplasmic compartment of E. coli.
Consequently, to further investigate this phenomenon, the fermentation broth was subjected to centrifugation, followed by fragmentation, and subsequent analysis using SDS-PAGE.
Fig. 9 SDS-PAGE of the LSPETase and LSPETase fused with the signal peptide after induction at 20 ℃ for 19h (M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of OmpA-LSPETase slurry; lane 4: Precipitate of OmpA-LSPETase slurry; lane 5: Supernatant of DsbA-LSPETase slurry; lane 6: Precipitate of DsbA-LSPETase slurry.)
The molecular weight of LSPETase is 30.2 kDa, while DsbA-LSPETase and OmpA-LSPETase are 32.3 kDa and 32.2 kDa, respectively.
As shown in Fig. 9, the soluble expression of LSPETase enzyme protein after the addition of the signal peptide was greatly enhanced. The comparison of the two signal peptides revealed that DsbA significantly reduced the insoluble expression of LSPETase enzyme in the precipitate, and the effect of OmpA, although it also reduced the insoluble expression of LSPETase enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPETase in the supernatant was significantly higher than that of LSPETase and DsbA-LSPETase. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPETase enzyme by increasing the soluble expression of LSPETase enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPETase enzyme. Both signal peptides were effective in soluble expression of LSPETase.
Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analyzed as follows:
Fig. 10 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the signal peptides.
Fig. 11 Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the signal peptides
An analysis of Figure 10 and Figure 11 showed that under the specified induction conditions, the protein solubility of DsbA-LSPETase and OmpA-LSPETase was increased by 1.7-fold and 1.4-fold respectively compared to the original LSPETase. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides effectively facilitates the solubility of the target protein.
In the field of synthetic biology, the selection of an optimal system requires careful consideration of multiple factors. To overcome the limitations associated with the current approach, our research team has developed a system for enhancing the soluble expression of the LSPETase enzyme in E. coli by incorporating two N-terminal signal peptides, namely DsbA and OmpA. Despite these efforts, the obtained results were not sufficiently significant and the operational process remained relatively cumbersome. Therefore, our objective is to identify a simplified approach that can substantially improve the expression level of the LSPETase enzyme.
In light of this, we conducted a thorough investigation of the existing literature and discovered the concept of molecular chaperones. By harnessing chaperone proteomes in individual plasmids, these chaperones collaborate to facilitate protein folding, thereby increasing the recovery rate of soluble proteins. This promising method offers the potential to enhance the expression of the LSPETase enzyme while ensuring ease of use.
In our experiments we need a protein that can assist in the correct folding of proteins. Certain protein molecules in the cell recognise polypeptides being synthesised or partially folded polypeptides and bind to certain parts of the polypeptide, thus helping in the translocation, folding or assembly of these polypeptides; this class of molecules is not involved in the formation of the more final product itself, and is therefore called molecular chaperones[3].
The Chaperone Plasmid Set, a chaperone proteome, was selected to construct a new plasmid by accessing the target proteins, and the chaperone proteomes act synergistically and participate in protein folding together, which can increase the recovery rate of soluble proteins.
We chose the Chaperone Plasmid Set for the chaperone proteome, and selected different chaperone plasmids, pTF16, pKJE7, and pGro7. We introduced the dual plasmid, pET-22b-LSPETase plasmid and molecular chaperone-expressed plasmid, into E. coli, and the recovery of soluble proteins can be increased through the synergistic effect of each chaperone proteome and the joint participation in protein folding.
Pairs of strains modified with were named pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase.
The procedure is the same as the above two experimental protocols: linearisation of the vector and amplification of the fusion-tagged DNA fragments by reverse PCR. After homologous recombination of the linearised vector and the amplified fragments, heat-stimulated transformation was performed. The transformed bacteria were cultured for induced expression. The expressed proteins were subjected to SDS-polyacrylamide gel electrophoresis to analyse whether the bands were correct or not, and to verify the validity of the experiment.
Fig. 12 SDS-PAGE of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase (M: maker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of pTF16/LSPETase slurry; lane 4: Precipitate of pTF16/LSPETase slurry; lane 5: Supernatant of pKJE7/LSPETase slurry; lane 6: Precipitate of pKJE7/LSPETase slurry; lane 7: Supernatant of pGro7/LSPETase slurry; lane 8: Precipitate of pGro7/LSPETase slurry.)
As depicted in Figure 12, the soluble expression of LSPETase enzyme after the addition of all three molecular chaperones was significantly enhanced, and the comparison of the three molecular chaperones revealed that pGro7/LSPETase was the most effective among them.
Further analysis of the protein gel was conducted to quantify the increase in protein expression using optical density measurements. The results were shown in Figure 13 and 14. Compared with LSPETase, the soluble protein expression levels of pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase were increased by 2.3-fold, 1.8-fold, and 2.2-fold, respectively. The conclusion demonstrates that the addition of molecular chaperones significantly enhances the solubility of the target protein.
Fig. 13 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase
Fig. 14 Percentage comparison of optical density analysis data of supernatant and precipitated protein bands of cell breakage solution of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase
In conclusion, the above three different strategies for enhancing soluble expression laid the foundation for the subsequent efficient soluble expression of PETase enzymes.
Ideonella sakaiensis[4] is a novel bacterium that can use polyethylene terephthalate (PET) as the main energy and carbon source, and it produces the enzyme IsPETase with PET hydrolysis activity. After a lot of literature research[5], we realized that mutation is a common method to increase the activity of IsPETase. Therefore, we decided to adopt the mutation strategy to improve the activity of IsPETase. By analyzing the structure and mechanism of IsPETase protein (Fig. 15), we selected 10 mutation sites,namely IsPETaseS93_I94insE,IsPETaseT116R,IsPETaseQ119F,IsPETaseS121E,IsPETaseQ126L,IsPETaseM157A,IsPETaseM157S,IsPETaseW159H,IsPETaseW185F and IsPETaseA240_S242del.
Fig. 15 Tertiary structure of IsPETase
Table.1 Reasons for selection of mutation sites
We designed primers for point mutation and amplified the pET22b-IsPETase plasmid as a template to obtain the single-point mutants (Fig. 16).
Fig. 16 Mapping of point mutant plasmid
The PCR system for the mutation was given in [Experiment], and 5 µL was taken for nucleic acid gel electrophoresis verification after PCR was completed. The rest of the system was demethylated with DpnI and then transformed into E. coli BL21 (DE3) sensory state. Single colonies were picked from the colonies grown after transformation and colony PCR was performed. Fig.17 shows the colony PCR bands verified by nucleic acid gel electrophoresis. After nucleic acid electrophoresis verification, the corresponding single colonies with correct bands were transferred to an LB (AMP) liquid medium, and sequencing verified that all 10 mutant plasmids had been successfully constructed.
Fig.17 Nucleic acid gel electrophoresis for PCR of colonies
Ten mutant strains were subjected to activation, expanded culture, and a series of protein purification operations to extract the target proteins using the method in [Experiment]. The volume of purified enzyme solution required for the 500 µL reaction system was determined based on protein concentration as in [Experiment]. The reaction with PET powder was carried out at 30℃, 37℃, and 45°C for 48 h, at the end of which the products were analyzed using High Performance Liquid Chromatography (HPLC).
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Fig. 18 Concentrations of the products TPA, MHET of 500 nM WT, S93_I94insE, T116R, Q119F, S121E, Q126L, M157A, M157S, W159H, W185F, and A240_S242del reacted with PET powder at different temperatures for 48h. (A) and (B) are the product concentrations obtained at a reaction temperature of 30℃; (C) and (D) are the product concentrations obtained at a reaction temperature of 37℃; (E) and (F) are the product concentrations obtained at a reaction temperature of 45℃.
As shown in Fig.18, IsPETaseS93_I94insE,IsPETaseT116R,IsPETaseQ119F and IsPETaseW159H product concentrations were higher than that of WT under the same conditions, indicating that the four mutants were more effective in degrading PET powders compared with WT.
At 30°C, the product concentrations of IsPETaseS93_I94insE and IsPETaseQ119F products were about 2 times that of WT, and IsPETaseW159H was about 2.5 times that of WT. Thus at 30°C, IsPETaseW159H had the most significant increase in enzyme activity and the best degradation of PET powder.
At 37°C, IsPETaseS93_I94insE,IsPETaseT116R, and IsPETaseQ119F were all elevated, but the effect was not significant. The concentration of IsPETaseW159H product was about 2.5 times that of WT. Therefore at 37°C, it was also IsPETaseW159H enzyme activity that was most significantly elevated.
At 45°C, the IsPETaseS93_I94insE product concentration was about 9 times that of WT, while IsPETaseW159H was about 5 times that of WT, which indicated that IsPETaseS93_I94insE is more adapted to higher temperatures than IsPETaseW159H, and this mutation site likely improves the enzyme's heat resistance.
Because IsPETaseW159H has the most obvious enhancement effect relative to WT, we decided to use IsPETaseW159H as a template in the later experiments, on which we superimposed S93_I94insE, T116R, and Q119F, respectively. we expected to obtain mutants with higher activity through superimposed mutations.
Similar to [Design 1], primers designed for previous single point mutations can be used directly due to the large spacing between the mutation sites of S93_I94insE, T116R, Q119F W159H, and W159H.
The same method as [Build 1] was successfully constructed for IsPETaseS93_I94insE/W159H, IsPETaseQ119F/ W159H. Multiple attempts to construct IsPETaseT116R/ W159H plasmid were unsuccessful, we will optimize the construction method later and continue the experiment.
Same method as [Test 1].
Fig. 19 Concentrations of the products TPA, MHET of 500 nM WT, S93_I94insE/W159H, and Q119F/W159H reacted with PET powder at different temperatures for 48h. (A) is the concentration of the products obtained at a reaction temperature of 30℃; (B) is the concentration of the products obtained at a reaction temperature of 37℃; (C) is the concentration of the products obtained at a reaction temperature of 45℃.
As depicted in Fig.19, the product concentration of IsPETaseQ119F/W159H was much higher than that of WT under the same conditions, whether at 30℃, 37℃ or 45℃, while the effect of IsPETaseS93_I94insE/W159H was much lower than that of WT, suggesting that simultaneous mutation of both the S93_I94insE and W159H sites reduces the enzyme activity.
At 30°C, the concentration of IsPETaseQ119F/W159H product was about 4 times that of WT; at 37°C, the concentration of IsPETaseQ119F/W159H product was about 5 times that of WT; at 45°C, the concentration of IsPETaseQ119F/W159H product was about 23 times that of WT. These indicate that the stacking mutant IsPETaseQ119F/W159H has much improved high temperature resistance compared with WT, but the activity of this mutant is higher at 37°C compared with 45°C. Learn2
We found that the enzyme activity of the IsPETaseQ119F/ W159H stacking mutant was significantly higher than that of WT, in agreement with the results of our model analysis. The strategy of using mutation to increase the activity of IsPETase essentially changes the structure of the protein, in addition to this, we also consider changing the extrinsic properties to increase the activity of the enzyme.
The hydrophobicity of the PET surface prevents IsPETase from binding to PET, which prevents IsPETase from functioning well. Therefore, we would like to add a hydrophobic domain, CBM, to IsPETase, so that under the force of water molecules, IsPETase associated with the hydrophobic domain will be easier to bind to the PET surface.Through literature research[6], we selected the hydrophobic carbohydrate domain CBM3, as well as a series of carbohydrate domains CBM4, CBM11, LSChi4CBM and LSChi5CBM found in our laboratory preservation cultures.
Fig. 20 Schematic diagram of the role of hydrophobic structural domains
Five recombinant plasmids were constructed by attaching the five structural domains mentioned above after IsPETase. However the direct fusion of IsPETase and CBM may lead to protein expression problems, so we added a linker when designing the primers, and the linker flexibly combines IsPETase and CBM when constructing the plasmids, as shown in Fig.21.
Fig. 21 Schematic diagram of the reorganization element
We respectively constructed recombinant plasmids with the structural domains of CBM3, CBM4, CBM11, LSChi4CBM and LSChi5CBM. IsPETase was first cloned from the pET22b-IsPETase plasmid. CBM3, CBM4, CBM11, LSChi4CBM, and LSChi5CBM were cloned from the plasmids kept in the laboratory of the research group, and then IsPETase was respectively fused with the five structural domains mentioned above. The pET-22b (+) plasmid was used as a template, and the linear vector was obtained by double digestion with NdeI and XhoI. Five recombinant plasmids were obtained by ligating each of the above five fusion proteins to the linear vector using the Gibson assembly method. The plasmids were transformed into E. coli BL21 (DE3) sensory state and single colonies were picked out from the colonies grown after transformation. The T7 promoter and the post-primer of the amplified structural domain were used for colony PCR, as Fig.22.
Fig. 22 Nucleic acid gel electrophoresis for colony PCR.(M: DL2000 Plus DNA Marker,1-5: pET22b-IsPETase-linker-CBM3,6-9: pET22b-IsPETase-linker-CBM4,10-14: pET22b-IsPETase-linker-CBM11,15-19: pET22b-IsPETase-linker-LSChi4CBM,20-22: pET22b-IsPETase-linker-LSChi5CBM.)
After nucleic acid gel electrophoresis verification, the corresponding single colonies with the correct bands were transferred to an LB(AMP) liquid medium and then sequenced to verify that the five recombinant plasmids were successfully constructed. The five recombinant plasmids were successfully constructed.
The above five recombinant engineering strains were subjected to activation, expanded culture, and a series of protein purification operations to obtain the target proteins using the methods in [Experiment]. The supernatant after cell lysis, the precipitate after cell lysis, and the purified protein was kept for SDS-PAGE analysis (Fig. 23).
Fig. 23 SDS-PAGE plots of supernatant, precipitated, and purified proteins of WT, IsPETase-CBM3, IsPETase-CBM4, IsPETase-CBM11, IsPETase-LSChi4CBM, and IsPETase-LSChi5CBM.(M: 180 kDa Prestained Protein Marker,1-3: Supernatant, precipitated, and purified enzyme solution of WT (protein size ~29kDa),4-6: Supernatant, precipitated, and purified enzyme solution of IsPETase-CBM3 (protein size ~46 kDa),7-9: Supernatant, precipitated, and purified enzyme solution of IsPETase-CBM4 (protein size ~39 kDa),10-12: Supernatant, precipitated, and purified enzyme solution of IsPETase-CBM11 (protein size ~39 kDa),13-15: Supernatant, precipitated, and purified enzyme solution of IsPETase-LSChi4CBM (protein size ~39kDa),16-18: Supernatant, precipitated, and purified enzyme solution of IsPETase-LSChi5CBM (protein size ~39kDa))
SDS-PAGE analysis (Fig. 23) showed that most of the target proteins were precipitated after ligating IsPETase to the structural domains. To solve the problem of protein insolubility, we decided to introduce the five constructed recombinant plasmids and the most effective molecular chaperone identified in [soluble expression] section into E. coli BL21(DE3). The supernatant of cell lyses, precipitates from cell lyses, and purified proteins were analyzed by SDS-PAGE.
The five recombinant plasmids that were successfully constructed in [Construct 1] and recombinant IsPETases were respectively introduced into E. coli BL21 (DE3) with molecular chaperone pGro7.
Fig. 24 SDS-PAGE plots of supernatant, precipitated, and purified proteins of WT, IsPETase-CBM3, IsPETase-CBM4, IsPETase-CBM11, IsPETase-LSChi4CBM, and IsPETase-LSChi5CBM after the introduction of the molecular chaperone pGro7.(M: 180 kDa Prestained Protein Marker,1-3: Supernatant, precipitated, and purified enzyme solution of WT (protein size ~29kDa),4-6: Supernatant, precipitated, and purified enzyme solution of IsPETase-CBM4 (protein size ~39 kDa),7-9: Supernatant, precipitated, and purified enzyme solution of IsPETase-CBM3 (protein size ~46 kDa),10-12: Supernatant, precipitated, and purified enzyme solution of IsPETase-CBM11 (protein size ~39 kDa),13-15: Supernatant, precipitated, and purified enzyme solution of IsPETase-LSChi4CBM (protein size ~39kDa),16-18: Supernatant, precipitated, and purified enzyme solution of IsPETase-LSChi5CBM (protein size ~39kDa).)
As shown in Fig.24, although there were bands in the precipitation, both the supernatant and the purified enzyme solution showed bands in the correct position. It indicates that our recombinant proteins were successfully expressed and purified.
Test 2
The purified protein concentration was recorded in [notebook], and the amount of purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration as described in [Experiment]. The reaction was carried out with PET powder at 30°C, 37°C, and 45°C for 48h, after which the products were analyzed by HPLC.
Fig. 25 500nM of WT, IsPETase-linker-CBM4, IsPETase-linker-CBM3, IsPETase-linker-CBM11, IsPETase-linker-LSChi4CBM, IsPETase-linker-LSChi5CBM at different temperatures. The products TPA and MHET concentrations reacted with PET powder for 48h. (A) is the concentration of the products obtained at a reaction temperature of 30°C; (B) is the concentration of the products obtained at a reaction temperature of 37°C; (C) is the concentration of the products obtained at a reaction temperature of 45°C.
As depicted in Fig.25, only the product concentration of IsPETase-CBM11 was higher than that of WT at 30°C and 37°C; the product concentration of both IsPETase-CBM4 and IsPETase-CBM11 was higher than that of WT at 45°C and IsPETase-CBM4 was about 4 times that of WT. It suggests that CBM4 enhances the hydrophobicity of the enzyme and may also enhance the high-temperature resistance of the enzyme. The results also showed that CBM11 enhanced the hydrophobicity of the enzyme at all three temperatures.
CBM4 was less active than WT at both 30 and 37°C, but CBM4 was more active than WT and CBM11 at 45°C. This indicates that CBM11 has higher enzyme activity than WT at both low and higher temperatures, and CBM4 has higher enzyme activity than WT at higher temperatures. Among the superimposed mutations, our results showed that IsPETaseQ119F/ W159H had the highest activity at 37°C; CBM11 hydrophobicity was best at 37°C after experimental screening at different temperatures. We expect to obtain a more active enzyme by ligating IsPETaseQ119F/ W159H to CBM11.
Connect IsPETaseQ119F/ W159H to CBM11 according to [Design 1].
The pET22b-IsPETaseQ119F/ W159H - CBM11 recombinant plasmid was successfully constructed. The method is the same with [Build 1].
The volume of purified enzyme liquid required for the 500 µL reaction system was determined based on protein concentration as in [Experimental]. After reacting with PET powder for 48 h at 37°C, we also performed the enzymatic reaction using PET film and industrial filter cloth as substrates. Since PET film and industrial filter cloth are more difficult to degrade due to their higher crystallinity than PET powder, we expected to use the most effective enzyme to attempt degradation. The products were analyzed using HPLC at the end of the process.
Fig. 26 Concentrations of the products TPA, MHET of the reaction of 500nM IsPETaseQ119F/ W159H - CBM11 with PET powder, PET film, and filter cloth at 37°C for 48h.
As shown in Fig.26, IsPETaseQ119F/ W159H - CBM11 degraded PET powder at about 6 times the product concentration of WT, degraded PET film at about 5 times the product concentration of WT, and degraded filter cloth at about 6 times the product concentration of WT. However, it is clear that IsPETaseQ119F/ W159H - CBM11 is much more capable of degrading PET powder than PET film and filter cloth.
After 48h degradation, we observed the changes on the surface of the PET film under an electron microscope(Fig. 27).
Fig. 27 (A) SEM images of PET films degraded by the enzyme-free system for 48h; (B) SEM images of PET films degraded by IsPETase for 48h; (C) SEM images of PET films degraded by IsPETaseQ119F/ W159H - CBM11 for 48h.
As depicted in Fig.27 and 28, the degree of degradation of the PET material can be reflected by the degree of surface depression in the SEM image. Since (A) was only immersed in buffer solution for 48h without degradation by enzyme addition solution, it presents a smooth surface in the SEM image. (B) is the effect after 48h of WT degradation, a slight depression can be seen on the surface, but the degree of depression is not obvious. (C) is the effect of IsPETaseQ119F/ W159H - CBM11 after 48h of degradation, it can be seen that some areas of depression are more obvious than the effect of WT.
Fig. 28 (A) SEM images of the degradation of industrial filter cloth by the enzyme-free system for 48 h; (B) SEM images of the degradation of industrial filter cloth by IsPETase for 48 h; (C) SEM images of the degradation of industrial filter cloth by IsPETaseQ119F/ W159H - CBM11 for 48 h. The degradation degree of the PET material can be reflected by the roughness of the surface in the SEM images.
Due to the high crystallinity of PET films and industrial filter cloths, IsPETaseQ119F/ W159H - CBM11 degrades PET films and industrial filter cloths much less effectively than it degrades PET powders. Therefore, it is necessary to pre-treat PET film and industrial filter cloth to reduce their crystallinity. We will optimize the experimental protocol and find a suitable pre-treatment method in the following experiments.
We will continue this experiment to screen more structural domains and stack more effective mutation sites to obtain more active enzymes. We hope our research can contribute to PET degradation and will continue to work on this in the future.
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