Latest revision as of 23:11, 21 October 2021

3xHA, MocloMania B2

This basic part codes for a 3xHA epitope tag that can be used for western blot detection of your fusion protein. It is based on the HA epitope tag first established in 1988 by J. Field and colleagues which is derived from a small segment of the viral protein hemagglutinin (HA), one of three integral membrane proteins in the Influenza A virus.^[1],^[2] Fusing your protein of interest with a 3xHA-tag enables its detection using inexpensive commercial primary antibodies against the HA epitope and thus eliminates the need for expensive protein-specific antibodies.

size 3.56 kDa

function antibody detection tag

amino acid sequence YPYDVPDYA GS YPYDVPDYA GS YPYDVPDYA

cloning position B2

plasmid backbone pAGM1276

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.

While L0_3xHA_B2 has been used to assemble several different L1 constructs, only one of them, L1_3xHA_RBD_mCerulean, has been transfected into Leishmania and showed significant protein expression after <b>immunostaining on western blot, see Figure 3.

When looking at construct 4 | L1_3xHA_RBD_mCerulean, a single protein band can be detected in the cell lysate P+. Taking into consideration any gel deficiencies influencing electrophoresis, this band can be attributed to the full-lenght fusion protein calculated to weigh about 55.7 kDa. Since no secretion tag was included in the construct, protein expression was exclusively observed within the cell lysate. So far, no immunoblot staining against the HA epitope has been conducted. Thus, the functionality of L0_3xHA_B2 could not yet be definitively verified. We are working hard on realizing this experiment in order to qualitatively validate the functionality of this basic part.

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.^[3] 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.^[4] 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 the part that you are looking at integrates into our part collection. See you there!

Reference Literature

1. ↑ J. Field, J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I. A. Wilson, R. A. Lerner, M. Wigler: Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. In: Molecular and cellular biology. Band 8, Nummer 5, Mai 1988, S. 2159–2165, PMID 2455217, PMC 363397
2. ↑ White JM, Hoffman LR, Arevalo JH, et al. (1997). "Attachment and entry of influenza virus into host cells. Pivotal roles of hemagglutinin". In Chiu W, Burnett RM, Garcea RL (eds.). Structural Biology of Viruses. Oxford University Press. pp. 80–104
3. ↑ 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.
4. ↑ 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