Latest revision as of 21:51, 21 October 2021

SARS-CoV-2 spike protein receptor binding domain, MocloMania B3

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

plasmid backbone pICH41258

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 L1 construct, proving its correct adaptation towards MoClo assembly, see Figure 2.

Several L1 constructs have been assembled with this part have been successfully transfected into Leishmania and resulting tagret protein expression could be observed after purification and immunoblotting, see Figure 3. 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 variety of different L1 constructs. For this, L0_RBD_B4 was usually fused to the sAP secretion tag and a C-terminal fluorescence marker as well as a 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 and 4.

In all of the blots shown above, the constructs carrying L0_RBD_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, L1_sAP_RBD_mVenus_GST and L1_sAP_RBD_mCerulean_GST showed significant purification results, see Figures 5 and 6.

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