Introduction - Design of the constructs for the MoClo library
The goal of our project was to use the Modular Cloning system (MoClo) to assemble gene constructs and introduce them into Leishmania tarentolae for protein production. For this, we created a library of 40 MoClo constructs for iGEM, consisting of one level-1 expression vector, 19 level-0 parts, and 20 level-1 constructs (the most important ones are shown in Figure 1). Constructs were propagated in TOP10 Escherichia coli, as this strain allows efficient cloning and high plasmid replication. The final level-1 constructs were transfected into Leishmania tarentolae. For this purpose, the Leishmania host strain T7-TR from Jena Bioscience was used, which belongs to the family of inducible LEXSY expression vectors. The pLEXSY_I-blecherry3 expression vector was domesticated for our L. tarentolae MoClo System. The finished expression vector was then used together with the respective level-0 parts to assemble level-1 constructs. These were cut with the restriction enzyme SwaI for transfection and integration into the Leishmania genome.
As a protein of interest, we chose the SARS-CoV-2 receptor binding domain (RBD). Because Leishmania tarentolae shows human-like glycosylation patterns, constructs with a sAP1 (secreted acid phosphatase 1) secretion signal were assembled such that the produced RBD is transported out of the cell into the medium. Secretion is necessary for glycosylation. Constructs without a secretion signal were built to demonstrate that the protein can also be produced in the cytosol of Leishmania where it is not glycosylated. The different tags selected for detection and purification were used to make the MoClo library as diverse as possible.
Domestication of vector
The first step for creating a functional expression system was to find a suitable expression vector that would allow for insertion of the desired gene constructs and efficient transfection into Leishmania tarentolae. For this, we relied on the “LEXSinduce3 Expression Kit”, distributed by Jena Bioscience GmbH, which is specifically designed to be used for heterologous protein expression in Leishmania. The vector contained in the kit is named pLEXSY_I-blecherry3 and carries a bleomycin resistance marker for selection as well as mCherry fused to the marker for the visualization of transfected cells . In order to utilize pLEXSY_I-blecherry3 as a plasmid backbone for Modular Cloning, any pre-existing BsaI recognition sites had to be removed from the plasmid to avoid fragmentation of the backbone during cloning. This was done by introducing single point mutations into the sites via PCR. New BsaI recognition sites at the 5’ and 3’ ends of the resulting PCR fragments should allow for their easy reassembly into a fully domesticated plasmid backbone via a standard MoClo reaction.
Figure 2 shows the plasmid map of our expression vector weird_plex. It contains all the necessary features for serving its purpose in our MoClo system, with an ampicillin resistance gene for transformation and propagation of the plasmid in E. coli, a lacZ-alpha gene cassette for blue-white screening in E. coli, a bleomycin resistance marker for selection of transfected L. tarentolae cells as well as a mCherry gene enabling monitoring of expression after induction.
The domestication of pLEXSY_I-blecherry3 proved to be far more difficult than initially anticipated. In numerous attempts of PCR amplification, one of the fragments repeatedly turned out to be flawed, hindering successful creation of the final vector. After several unsuccessful attempts at amplifying the desired three PCR fragments, we decided to divide the problematic fragment into several smaller parts. Further attempts with new primers revealed a more detailed view onto the problem. After identifying a problematic 400-bp sequence spanned by primers plex-400-rev and plex-2-for, we decided on trying to amplify the sequence again which led to promising results, as shown in Figure 3.
Analysis of the underlying DNA sequences revealed a highly repetitive region (Fig 2A; labelled FU) within the fragment in question, suggesting possible difficulties of Q5 DNA polymerase with replicating them. Sequencing showed that there were about 150 bp missing in that fragment.
Figure 4 shows the sequencing results that revealed the missing 150 bp containing a highly repetitive region between bp 1990 and bp 2030.
We tried to amplify the sequence from another template and for this used the pLEXSY_IE-blecherry4 expression vector from Jena Bioscience GmbH. The 400 bp fragment, which had to be amplified out of the plasmid, was homologous to the sequence on our destination vector.
Figure 5 shows the DNA amplified by PCR with the different templates. It can be seen, that the previous version of our domesticated vector (lane 2) is missing base pairs, which seem to be present in the PCR amplification with the other template. Hence, we used this fragment for further ligation attempts.
Unfortunately, we could not completely solve the problem, as sequencing revealed that there were still about 30 bp missing from the plasmid. Thus, a new strategy was employed, relying on cutting out the fragment covering the problematic region from the original pLEXSY_I-blecherry3 and inserting it into the otherwise domesticated vector.
In order not to re-introduce any BsaI recognition sites, only a specific set of restriction enzymes could be used for this. One of them was ClaI, which is blocked by dam-methylation. In order to bypass this issue, the relevant DNA fragments were transformed into CGSC5127, a dam- E. coli strain, followed by plasmid miniprep and digest with ClaI and SpeI.
Figure 6 shows the result of the digest with ClaI and SpeI. Lanes 4 and 8 show the expected band patterns of the double digest. Bands where extracted from the gel for further experiments. The ensuing ligation brought out a correctly assembled plasmid, which could be verified by restriction digest as well as sequencing (Fig. 3B) to be the successfully domesticated and intact weird_plex vector.
Another important step in establishing the expression vector for our MoClo system was the introduction of a lacZ-alpha gene cassette into the plasmid, which allows for blue white screening after cloning and transformation into LacZ-omega carrying E. coli cells. Along with the lacZ-α gene cassette, new BsaI restriction sites were introduced into the plasmids that serve as entry site for MoClo assemblies.
Figure 7 shows the successful amplification of the lacZ-alpha gene cassette originating from pICH47742 (plasmid backbone from the Weber collection). After amplification, the lacZ fragment as well as the domesticated destination vector were sequentially digested with restriction enzymes BglII and NotI and ligated with T4 DNA ligase, leading to the finished and ready-to-use expression vector weird_plex.
Figure 8 shows a test digest of the original vector pLEXSY_I-blecherry3 compared to the domesticated expression vector weird_plex. The digest along with sequencing of the whole plasmid show definitive proof of a fully assembled and domesticated expression vector containing all important features for our MoClo System in Leishmania tarentolae.
Cloning of parts
To build a library of modular parts, we wanted to design a number of different fluorophores, affinity tags and signal peptides. Eventually, we had to cut down the number of parts, since the domestication of our destination vector turned out to be more time consuming than expected. Thisis our final list of constructs.
Some of the parts we have were acquired via de novo synthesis, where we optimized the codon usage for Leishmania. Here you can find out how to create a MoClo part. Some parts were made via PCR using the MoClo collection for Chlamydomonas reinhardtii as templates.
Our sAP secretion signal was accquired using PCR on the pLEXSY_I-blecherry3, which is our original vector. Successful PCR amplificates were cloned into the respective target vector using the BbsI restriction sites. The MoClo constructs where then transformed into E. coli and selected via blue-white screening.
All acquired L0 parts were test digested and sequenced to confirm the correct insertion of the part into the vector. Correct plasmids were propagated in TOP10 E. coli and isolated with plasmid extraction.
For creating the L1 constructs, we performed overnight MoClo reactions with BsaI and transformed the resulting plasmids in TOP10 E. coli. Vectors with an insert were identified via blue-white screening. Plasmids were propagated in E. coli and test digested:
Upon successful assembly of our desired gene construct, the plasmid was almost ready for transfection into Leishmania.
The insertion into the Leishmania genome works better if the plasmid is linearized before transfection. We achieved this through digesting the plasmid with SwaI, which cuts the plasmid twice to remove the E. coli replication cassette (1957 bp).
To increase transfection efficiency of Leishmania, a large amount of plasmid DNA (about 5 µg) is needed. Since we lose a little bit of DNA during every step, we linearized 10 µg of DNA using SwaI. The Leishmania cassette was extracted from the gel and handed over to the Leishmania team.
Transfection of Leishmania
To finally transfect our level-1 constructs into Leishmania, we transfected usually 108 cells with 5-10 µg DNA using a nucleofector. After transfection, the cells were transferred to BHI medium, incubated over night at 27 °C and plated onto BHI agar containing the selection antibiotic bleomycin.
The BHI agar plates were incubated at 27 °C until the colonies reached a diameter of one to two millimeters, which were usually visible after one to two weeks. The colonies were then transferred into liquid medium and induced with tetracycline after three to four days of growth time.
To ensure that the integration of our plasmid into the genome was successful, we performed a colony PCR using primers binding to the blecherry gene.
The second lane was loaded with the reaction mixture containing the pLEXSY_I-blecherry3 culture. The transfected construct carries the blecherry gene and therefore a band at 349 bp should be detectable on the agarose gel. As seen on Figure 13 you can see the agarose gel, one band in the pLEXSY_I-blecherry3 (Fig.13) construct was visible at the expected size of approximately 340 bp.
Since previous transfection had grown cultures on the negative control, we wanted to make sure that our LEXSY original cell culture did not carry bleomycin resistance.
However, since no band was seen on the agarose gel in the original LEXSY cell culture (Fig. 13), it can be assumed that the Blecherry marker entered the Leishmania through transfection and that our transfections are accordingly successful.
The second negative control, Lt UCAS pTB007, was used because it was a culture transfected with a different vector that did not contain the blecherry marker. This way we could be sure that our primer binds to the correct sequence and prove that our transfection was successful.
Detection of mCherry production in Leishmania tarentolae
To detect the production of the protein mCherry, which is produced in fusion with the bleomycin binding protein and encoded by the expression vector, the spectrum of the fluorescent protein was measured in cells harboring different constructs. mCherry has its emission maximum at a wavelength of 610 nm and its extinction maximum at 587 nm . We also measured uninduced cultures in order to analyze the effect of induction with tetracycline. The addition of tetracycline leads to binding of the tetracycline to the Tet-repressor and thus detachment of the repressor from the operator. Hence expression of mCherry should be lower in uninduced cultures compared to induced cultures . Cells containing pLEXSY_I_blecherry3 (Fig.14A) and L1_sAP_RBD_3xHA (induced) (Fig.14B) have the highest emission (A: 348909 RFU, B: 345258 RFU) and extinction values (A: 315642 RFU, B: 356666 RFU). The uninduced cultures can not be compared with the induced cultures because they are not originating from the same colony. Hence it is possible that the uninduced cultures show higher expression of proteins than the induced cultures, as protein expression is variable between cultures. Expectedly, the negative controls and the BHI media do not show peaks at the maxima of both spectra. Nevertheless we can verify a successful transfection and the production of mCherry.
To demonstrate the functionality of our system, we needed to prove that our target proteins were produced by Leishmania. Therefore, a big step in our project was detecting the target proteins after transfection using Western blots.
For all blots, we withdrew cell culture containing 108 cells and separated them from the supernatant. The cells were immediately prepared with 5x Laemmli loading buffer diluted to a 1x concentration with 1x cOmpleteTM protease inhibitor including 2% 2-Mercaptoethanol, whereas the supernatant was precipitated with a final concentration of 10% trichloroacetic acid (TCA) and washed with 1 ml acetone priorly. The finished samples were loaded onto 15% SDS gels.
All blots show a significant increase in the amount of protein after the induction with tetracycline. Furthermore, it can be observed that the supernatant contains far more protein in all samples including sAP constructs and the RBD is cleaved off leading two double bands, whereas cells only show one band and constructs without sAP do not show bands in the supernatant as seen in 15A and 15B. To further validate the results we achieved with the RBD antibodies, we stained blots with HIS and GST antibodies.
Most of the bands lay higher than expected, which suggests an underlying technical problem, for example the percentage of the SDS gels at 15% being too high. But as the heights add up in relation to each other, we are still convinced that the results are valid, especially because we were able to confirm them with different antibodies.
Protein purification showed itself to be more difficult than we expected. After a lot of trials and errors, purifications using different affinity tags, trying a lot of different protocols, we finally were able to get some results.
To make use of recombinant proteins in medicine, research, or therapeutics you need to be able to purify the protein. We tried purifying our RBD from Leishmania supernatant via a Glutathion-Sepharose column, to which our GST-tagged RBD binds. The Western blot showed a clear signal for the GST-RBD (51.1 kDa) in our TCA precipitated eluate. Also, there seems to be RBD (25.1 kDa) in the washing steps, from which we concluded that our GST tag gets cleaved off to some extent.
We stained the same blot again with GST to confirm this (Figure 17.b). The signal for free GST was far stronger than that for the RBD-GST fusion protein (Figure 17.a). The purification process seemed to work, although it looks like we lost a lot of GST during the washing steps. The precipitated eluate also showed a RBD-GST signal (51.5kDa) as well as GST only (26.5 kDa). Therefore, we confirmed that the GST tag is partially cleaved off at the TEV cleavage site, which is located between the RBD and the GST.
The BCA assay resulted in a concentration of 0.13 mg/ml protein in the eluate. Which means in our total of 3 mL eluate we had 0.4 mg of our RBD-GST which comes down to 7.6 µg of protein in 1 mL of culture.
The GST purification left us with GST-tagged RBD. To purify only RBD we made use of the TEV cleavage site in between the RBD and the GST tag.
Apparently, the concentration of the RBD-GST fusion protein was too low to detect via Western blotting and only a faint band (51.1 kDa) was visible in input and eluate of after GST purification. The purification of GST was successful, although much GST remained in flow-through and wash fractions, although the amount of column material was calculated for the protein concentration that was determined via the BCA assay (Figure 18).
The purification shows large amounts of GST as well as of the RBD-GST in all fractions. P. supernatant and P. resuspended both show RBD-GST (51.5 kDa) as well as GST (26.4 kDa), indicating that not all of our protein was soluble after precipitation with ammonium sulphate.
We wanted to purify our RBD_mCerulean/mVenus using GST affinity chromatography to finally carry out our activity assay with RBD, where we could show the binding of the RBD to the ACE2 receptor via fluorescence of the fused mCerulean or mVenus. The signal of GST is visible in input, flow-through, wash 1, as well as in all eluate fractions. The eluate fractions show three signals. The first one (78.2 kDa) represents the entire fusion protein. All bands seem to run with a slightly larger apparent MW than expected, which could be due to glycosylations. The second band resembles mCerulean-GST/mVenus-GST. Here it appears that the RBD was cleaved off, pointing to presence of a protease that cleaves between the RBD and the fluorophore. The third band (26.4 kDa) represents free GST, which indicates another cleavage at the TEV recognition site.
Not only did we want to show that our protein gets expressed in the supernatant, or that it can successfully be purified through a GST-column, but that it also is functional. For this we chose two different methods. The activity of the RBD can be proven via Western blot, and with the right tag, through fluorescence microscopy.
For this method, we used human HEK 293T +ACE2 +TMPRSS2 (HEK+) cells that we got from our sponsor VectorBuilder. This specific cell line has a mutation that ensures an overexpression of ACE2 (angiotensin I converting enzyme 2), which is the protein on the surface of the human cells, where the RBD binds. As negative control we used HEK 293T cells that don’t express ACE2 (HEK-).
We applied the maximum volume that we had at our disposal and incubated the HEK-cells with our protein for an hour at 37°C and with 5% CO2. Following the incubation, we removed the medium and washed the cells with PBS and with that, all the residuals away. If our RBD successfully bound to the ACE2 protein on the cell membrane of the HEK-cells, it should have stuck, even after washing. Prior to loading the samples into SDS-gel wells, we lysed the cells with RIPA Buffer. On the blot we used primary antibodies against RBD (m α RBD 1:2000) and separately against ACE2 (m α ACE2 1:1000) with which we detected the specific proteins.
We incubated one well of HEK-cells with RBD_Strep8His and another well with weird plex (w_plex), where we only concentrated the supernatant from Leishmania through ammonium sulfate precipitation. One further well HEK-cells were incubated with RBD_TEV_GST which was purified through an GST column. All those samples were also incubated with HEK--cells that did not express ACE2 as negative controls.
For the protein RBD_Strep8His on HEK+ cells we can see a band at the right height, which is a first indication of functionality of our protein RBD. Furthermore, the band is not present in the negative control (HEK--RBD-Strep8His) where we incubated the HEK--cells with the same sample, which proves that the band is not a cross reaction of the antibody with any other proteins of the HEK-cells or Leishmania supernatant.
The sample with RBD_TEV_GST shows no such band. This could be because the RBD got lost during purification, or because the GST tag, which is as big as the RBD itself, interferes with the binding with ACE2. The HEK+-cells that were incubated with w_plex show, as expected, no RBD.
The following blot (Figure 23) was treated the same way as the one mentioned above (Figure 22), but with a few additional constructs. None of the added constructs showed a band at the expected molecular weight from RBD, but the potential activity of the sample RBD_Strep8His could be replicated with another set of HEK+-cells (Figure 23).
The band by the RBD_Strep8His sample is clearly visible, and no cross contamination of the antibodies could be detected in the negative control where no protein should be in the supernatant (w_plex) or nothing was incubated (HEK+). In addition to that, the RBD-GFP construct could be detected as a positive control for the specific binding of the antibody (Fig.23).
In Addition to the western blots, we looked at incubated HEK-cells with samples of RBD with a fluorescent tag through the microscope. The only difference to the cell preparation for blots, is an embedded coverglass in the 6-well-plate prior to sowing the cells. To visualize the nucleus, it was stained with DAPI solution added to a solution that extends the emission of the fluorophores (ProLong™ Glass Antifade Mountant).
The microscopy images (Figure 24) were from HEK+ and HEK--cells, incubated with RBD_mCerulean_TEV_GST that was purified through an GST column.
It is clearly noticeable that the HEK+-cells are mutants and therefore not as fit as the HEK--cells. The mCerulean fluorophore has the same excitation wavelength as DAPI, which makes it difficult to distinguish them from one another. Nevertheless, a slight shimmer around the HEK+-cells is visible, that is not apparent in the HEK--cells. That could be an indication for the binding of RBD onto the ACE2 on the cell membrane.
We also had another fluorophore tag mVenus, and incubated the sample RBD_mVenus with HEK- - cells and with HEK+ - cells (Fig. 25) The images showed ambiguous results. We can see yellow structures at certain areas at the cell membrane, but those are not very clear. This maybe could be a first indication for a further functional protein.
However, those experiments were only done once (Figure 23), or twice (Figure 24 and 25) and therefore should be considered as preliminary data. To definitively prove the functionality of our RBD, more replicates are needed. This work will be continued in the next months, and till then be hopefully ready for publication. More, and different kinds of activity assays are scheduled, like pull downs and immunoprecipitation, and immunofluorescence.
During the course of the year we were able to build a modular library of genetic parts suitable for Leishmania and domesticate the respective destination vector. With this vector and the library, we were able to successfully transfect L. tarentolae and detect our desired proteins. Although protein purification was more complicated than expected, we were able to purify the RBD, our protein of interest and even showed its functionality via an activity assay with human ACE2-cells.
For further improvement of the MoClo Mania system, the library has to be expanded, new parts e.g. more fluorophores or detection tags can be added, as well as more different purification tags to offer an even bigger variety of parts.
To improve the expression level of the constructs, there are a variety of possibilities. Via growth curve and protein quantification we want to find out when after adding tetracycline protein harvesting makes most sense and if its concentration impacts protein yield.
We also want to improve the purification of our protein and try out some more purification methods like HPLC or FPLC. We are also facing the problem that our TEV cleavage site gets cleaved in Leishmania, so we have to think about how to solve this problem. One idea was a more specific protease inhibitor mix during growth of Leishmania, as Dr. Christian Janzensuggested, as well as adding different protease inhibitors during purification itself. Another possibility regarding the cleavage of our fusion protein might be changing the TEV site to another cleavage site. Furthermore, we want to investigate the glycosylation capabilities of L. tarentolae using different comparison methods, e.g., mass spectrometry or reversed-phase chromatography.
Apart from the improvements on our library and the general project, we also want to further improve and test our example protein, the receptor binding domain (RBD) of Sars-CoV-2. We want to show the binding to human ACE2-receptor again, but via a fluorophore fused to the RBD e.g. L1_sAP_RBD_mVenus_TEV-GST.
We also want to further research on to the mechanism of the RBD and want to expand our work and not only express the RBD but the full length Sars-CoV-2 S protein. The S protein of the Sars-CoV-2 is a highly glycosylated protein which makes Leishmania a suitable expression host. Since the glycosylation pattern of the S protein plays a major role in binding to the human ACE2 receptor and therefore the immune response, our research could provide more insights into a possible protein based vaccine or ELISA based antibody tests.
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