PROOF OF CONCEPT
One of our primary goals was to ease the use of the organism Leishmania tarentolae for the production of pharmaceutically important proteins. We therefore decided to introduce the Modular Cloning system to Leishmania, which is based on Golden Gate Assembly.
For this it is important to note that each organism has its own codon usage. This means that if you want to express a protein in a particular organism, you need to adapt the DNA sequence of the gene encoding your protein of interest to the codon usage of that organism. Here you can find the codon usage of Leishmania tarentolae.
In our project, we mainly focused on the expression of the receptor binding domain (RBD) of Sars-CoV-2. This was fused to a secretion signal (sAP), different fluorophores (mVenus, mCerulean) and purification tags (Strep8His/GST).
The secretion signal is fused to proteins to achieve secretion of the desired protein into the medium and through this simplify the purification process. The fluorophores are used to detect the protein and the purification tag is necessary to purify the protein after successful secretion.
For proteins to be expressed in Leishmania, an expression vector coding for this protein must be transfected into Leishmania. Before, we had to modify the expression vector such that it was compatible with the MoClo system.
To this end, we had to remove BsaI recognition sites from the original plasmid pLEXSY_I-blecherry3 from Jena Biosciences. Since there were three BsaI sites in the original plasmid, we had to design six primers, forward and reverse, for each of the three sites. Each of these primers contained point mutations at the BsaI recognition site. PCR was used to amplify the altered fragments. Since sequencing showed that sequence stretches were missing in our final vector (they were extremely GC-rich), we decided to cut the erroneous sequence from the original vector and insert it into our domesticated vector. The problem with this, however, was that the only suitable restriction enzyme was dam methylation sensitive. Therefore, we first had to transform the vectors into a dam - E. coli strain. After purification of the plasmid DNA, we were able to cut the vector and insert the cut sequence into our domesticated vector.
A diagnostic digest was then used to demonstrate that the BsaI recognition sites were successfully removed. In addition, the LacZ gene was inserted using traditional cloning to enable blue-white screening.
Parallel to the domestication of our expression vector, we designed different L0 parts. It was important to pay attention to the correct codon usage and to adjust the overhangs for the correct cloning position. We obtained different parts either through designing the sequence and having it synthesized or, for smaller parts, we performed a PCR on parts existing for Chlamydomonas reinhardtii and simply adjusted the overhangs to fit our cloning position. Chlamydomonas has a similar codon usage as Leishmania tarentolae.
To clone a desired L0 part, it was ligated into a suitable destination vector using MoClo, which results also in deletion of the LacZ gene present in that vector. The enzyme BbsI was used for this purpose. The resulting vector was transformed into E.coli and selected by blue-white screening. An overnight culture was prepared from the positive (white) E.coli to produce sufficient plasmid. Plasmid DNA was then isolated and test-digested to verify correct cloning of the L0 part.
After successful construction of the vectors containing the L0 parts, L1 vectors combining various L0 parts were produced. For this purpose, the desired L0 parts and the destination vector were digested with the enzyme BsaI and ligated, with the specific overhangs allowing assembly of the parts in the desired order.
Subsequently, the obtained plasmids were transformed into E.coli. Using blue/white selection, colonies harboring successfully assembled L1 vectors were identified and grown in overnight cultures. After plasmid preparation, they were test-digested, again propagated in E. coli, purified, and given to the Leishmania team for transfection.
The agarose gel shows that PCR on DNA extracted from Leishmania cells transfected with pLEXSY_I-blecherry3 gives rise to a band at about 340 bp. Our primers were designed to bind to the Bleomycin resistance gene, which should result in a size of the amplified DNA of 349 bp. Another primer set was designed to bind to a gene in the genome, for which the amplified DNA is supposed to have a size of 920 bp. These results confirm successful transfection.
However, since this only confirms successful transfection, not successful protein production, we performed Western blots with proteins from cultivated Leishmania cells harboring our different constructs before and after induction with tetracycline.
The western blots show the signals expected for the RBD, which allows us to conclude that all transfections were successful.
Since we wanted to not only detect but also purify our protein, we grew cultures with larger volumes (50-500 ml) and induced them with tetracycline prior to affinity chromatography.
But not only were we able to purify our RBD_GST but also show that it probably is functional, because we did one successful activity assay.
The visible band for RBD 27 kDa shows that our RBD binds to the ACE2 receptor. In addition to the detection via Western blot, we could also show the binding of our RBD_mCerulean to the ACE2 HEK-cells via flourescence microscopy. Since there was only one try for those activity assays, this is only preliminary data!
Finally, we can see that our project to establish the MoClo system in Leishmania was successful. We were able to domesticate an expression vector for MoClo, build several L0 parts for secretion, detection and purification, to express proteins of interest in Leishmania, and then to purify them. Also our first tries of activity assays hint to a functionality of the RBD.