Latest revision as of 22:50, 21 October 2021

SARS-CoV-2 spike protein receptor binding domain, MocloMania B3_B4

This part codes for the receptor binding domain of the SARS-CoV-2 spike protein. These spike proteins are part of multimeric protein structures called spikes that project outwards from the virus envelope’s outer surface.^[1] As one of the four viral structural proteins it is essential for the recognition, binding and entry of the virus into the host’s cells.^[2] This is mediated by the receptor binding domain, located in the S1 region of the spike protein, due to its specific binding to the angiotensin-converting enzyme 2 (ACE2) located on the outer membrane of many mammalian and bird cells.^[3] This binding results in a TMPRSS2 mediated proteolytic cleavage of the spike protein S2 region, exposing a viral fusion peptide and thus enabling virus entry into the host cells.^[4]

size 25.1 kDa

function coding sequence

cloning position B3_B4

plasmid backbone pAGM1287

Data

We were able to successfully clone this basic part into its respective L0 plasmid backbone and to confirm the integrity of the L0 construct via restriction digest and gel electrophoresis, see Figure 1. Furthermore, we were able to include it into a variety of L1 constructs, proving its correct adaptation towards MoClo assembly, see Figure 2.

Since our project's objective was to test the efficiency of our MocloMania expression system by expressing and purifying recombinant SARS-CoV-2 receptor binding domain, this basic part was incorporated into a vast variety of different L1 constructs. For this, L0_RBD_B3_B4 was usually fused to the N-terminal sAP secretion tag and a variable C-terminal purification or detection tag. Most of these L1 constructs have been successfully transfected into Leishmania, resulting in recombinant protein expression that could be observed via immunostaining on western blot, see Figures 3-5.

In all of the blots shown above, the constructs carrying L0_RBD_B3_B4 were successfully stained with the help of anti-RBD antibodies. This verifies the correct recombinant expression of the underlying basic part sequence. In order to further analyse the quality of the expressed receptor binding domain, analysis blots were follwed by purification procedures to harvest produced RBD for downstream activity assays. So far, only L1_sAP_RBD_GST showed significant purification results, see Figure 6.

Here, the RBD_GST fusion protein was stained with anti-GST antibodies. This doesn't only visualize RBD_GST (expected around 52 kDa), but also singular GST protein expected at around 26 kDa. In order to read more on the interpretation of this band pattern as well as on the poor purification efficiency, please consult the L0_GST_B5 part page.

Despite very low purification yield, the samples resulting from GST-purification, along with other constructs carrying L0_RBD_B3_B4, were tested for functional activity of the receptor binding domain in an ACE2 binding assay. For this, we used human HEK 293T +ACE2 +TMPRSS2 (hek⁺) cells that we were given to us by our sponsor VectorBuilder. This specific cell line has a mutation that renders an overexpression of the RBD receptor angiotensin I converting enzyme 2, short ACE2. As negative control we employed HEK 293T cells that don’t express ACE2 (hek^-). For a specific protocol for the conduction of this activity assay utilizing human cell culture, please refer to our Experiments page.

For the activity assay seen in Figure 7, one well of HEK-cells was incubated with L1_sAP_RBD_Strep8His and another well with weird_plex,for both of which the cell culture supernatant was concentrated through ammonium sulfate precipitation. One further well of HEK-cells were incubated with the purified L1_sAP_RBD_GST. HEK^- -cells were incubated as negative controls.

For the fusion protein RBD_Strep8His on HEK⁺ cells, we can see a band at the expected height (27 kDa), 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. This proves that the band is not a cross reaction of the antibody with any other proteins of the HEK cells or Leishmania supernatant.

The purified RBD_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, structurally interferes with ACE2 binding. The HEK-cells that were incubated with w­eird_plex show, as expected, no RBD.

The MocloMania collection

This basic part is part of the MocloMania collection, the very first collection of genetic parts specifically designed and optimized for Modular Cloning assembly and recombinant protein expression in the protozoan parasite Leishmania tarentolae.

Are you trying to express complexly glycosylated proteins? Large antibody side chains? Human proteins that require accurate post-translational modification? Then Leishmania might be just the right organism for you! Leishmania tarentolae’s glycosylation patterns resemble those of human cells more closely than any other microbial expression host, while still delivering all the benefits of microbial production systems like easy transfection and cultivation.^[5] So instead of relying on mammalian cell lines, try considering Leishmania as your new expression host of choice!

Our MocloMania collection will allow you to easily modify your protein of choice and make it suitable for downstream detection and purification procedures - all thanks to the help of Modular Cloning. This cloning system was first established by Weber et al. in 2011 and relies on the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequence, hereby generating four nucleotide overhangs.^[6] Every basic part in our collection is equipped with a specified set of overhangs that assign it to its designated position within the reading frame. These so-called cloning positions are labelled B2-B5 from upstream to downstream. By filling all positions with the basic parts of your choice, you can easily generate variable genetic constructs that code for the fusion protein of your desire.

We furthermore provide a specifically domesticated Leishmania expression vector, named weird_plex, which will package your fusion construct into a functional transcriptional unit that is optimized for high expression in Leishmania.

The best part? Because of the type IIS restriction properties and the specifity of the generated overhangs, restriction and ligation of your construct can all happen simultaneously in a simple one-step, one-pot reaction. This will safe you a lot of time and frustration in your cloning endeavours!

Do we have your attention? In the table below you can find some basic information on how our cloning system, along with most other MoClo systems, is set up. Please feel free to check out our wiki to find more information on Leishmania and Modular Cloning as well as to understand how this basic part integrates into our part collection. See you there!

Reference Literature

1. ↑ Aronson JK (25 March 2020). "Coronaviruses – a general introduction". Centre for Evidence-Based Medicine, Nuffield Department of Primary Care Health Sciences, University of Oxford.
2. ↑ F. Li, Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 3, 237–261
3. ↑ Shang, J., Ye, G., Shi, K. et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221–224 (2020). https://doi.org/10.1038/s41586-020-2179-y
4. ↑ M. Hoffmann et al., SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e8 (2020).
5. ↑ Langer T, Corvey C, Kroll K, Boscheinen O, Wendrich T, Dittrich W. Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae. Prep Biochem Biotechnol. 2017 Nov 26;47(10):1008-1015. doi: 10.1080/10826068.2017.1365252. Epub 2017 Aug 31. PMID: 28857681.
6. ↑ Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. https://doi.org/10.1371/journal.pone.0016765
7. ↑ Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. https://doi.org/10.1371/journal.pone.0016765
8. ↑ Patron, N.J., Orzaez, D., Marillonnet, S. et al., (2015), Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol, 208: 13-19. https://doi.org/10.1111/nph.13532
9. ↑ https://www.addgene.org/cloning/moclo/, last visited 10/16/21, 19:00 ECT