Team:TU Kaiserslautern/Part Collection
THE MOCLOMANIA COLLECTION
FLUORESCENT TAGS
mVenus
size | 26.9 kDa
excitation wavelength | 515 nm
emission wavelength | 537 nm
available in cloning positions B3_B4, B4 and B5
The protein with the poetic name mVenus is a yellow fluorescent protein and a derivation of the famous green fluorescent protein GFP, originally isolated from the bioluminescent jellyfish Aequorea victoria in 1962. Even though the purpose of this extraordinary sea creature‘s bioluminescence is still unknown, its fluorescent proteins are of immense importance to modern biological research. This is not surprising, considering the great practicability of fluorescent reporter systems. By coupling a protein of interest with a fluorescent tag such as mVenus, the target protein can be easily and non-invasively detected within living cells as well as cell preparations with the help of fluorescence spectroscopy and microscopy.
mCerulean
size | 26.8 kDa
excitation wavelength | 435 nm
emission wavelength | 477 nm
available in cloning positions B3_B4, B4 and B5
Much like mVenus, the cyan fluorescent protein mCerulean is derived from the green fluorescent protein GFP originally isolated from the bioluminescent jellyfish Aequorea victoria in 1962. Like its molecular siblings, it can be cloned to a gene of interest and thus provide fast and non-invasive screening opportunities for the resulting fusion protein, for example via fluorescence spectroscopy or microscopy.
PURIFICATION TAGS
The 8His-tag is a so-called polyhistidine-tag, meaning that it is a small amino acid chain consisting of, in this case, eight consecutive histidine residues. The histidine sidechain contains a heterocyclic imidazole ring which is negatively charged in neutral to basic conditions and can coordinate with metal ions with very high affinity. Thus, equipping a target protein with a terminal histidine rich peptide sequence allows for purification of the protein via immobilized metal ion affinity chromatography. Here, divalent nickel or cobalt ions are attached to a carrier material and bound by the polyhistidine-tag upon addition of the cell lysate. Meanwhile, non-tagged proteins only display weak metal ion affinity and can be discarded in the flow through. Later on, the bound target protein can be easily eluted from the carrier material with the help of pH titration or high excess molecular imidazole which competes with the His tag for metal ion coordination. Since His tags are small in size and rely solely on the protein’s primary structure, they are the preferred choice for protein purification under denaturing conditions. The B3 version of this basic part is fused to an HRV 3C protease recognition site, whereas the B5 version is fused to a Strep-tag II.
When it comes to biomolecular detection and purification set-ups, the Strep-tag system is an absolute laboratory staple. In its principle it relies on the high affinity binding of the homo-tetrameric protein streptavidin, first isolated from the bacterium Streptomyces avidinii, to the vitamin biotin. The binding of these two biomolecules is one of the strongest non-covalent interactions observed in nature and inspired the creation of many derivatives with even further enhanced binding capabilities. The German biotech company IBA Life Sciences developed a synthetic eight amino acid peptide called Strep-tag II which binds to the specifically engineered streptavidin derivative Strep-Tactin with high specificity. Thus, fusing a gene of interest to a Strep-tag II allows for efficient purification of the target protein by affinity chromatography over immobilized Strep-Tactin. The bound target protein can later be eluted with desthiobiotin. Due to its small size and biochemical inertia, the addition of a Strep-tag usually doesn’t influence correct target protein folding and functionality. Furthermore, the purification procedure can be executed under physiological conditions, making the Strep-tag system particularly suitable for purification of native, functional proteins. The B3 version of this basic part is fused to an HRV 3C protease recognition site, whereas the B5 version is fused to an 8His-tag.
The GST-tag has been around since the late 1980s and, unlike most other purification tags, does not only consist of a small peptide, but rather an entire functional protein. GST is short for glutathione S-transferase, a highly abundant cytosolic enzyme that is usually involved in protecting prokaryotic as well as eukaryotic cells from extracellular toxins. It does this with the help of the redox peptide glutathione, to whose reduced form it binds with very high affinity. By fusing a gene of interest with a GST-tag, the resulting fusion protein can be purified from cell preparations with the help of affinity chromatography, employing reduced glutathione (GSH) as the respective column material. Due to the highly specific binding, only GST-tagged target protein should remain bound to the column and can afterwards be eluted by adding high excess molecular GSH. Since the affinity binding relies on the structural integrity of the GST protein, GST-tag purification can only be performed under native, non-denaturing conditions. The B3 version of this basic part is fused to an HRV 3C protease recognition site, whereas the B5 version is fused to a TEV protease recognition site.
DETECTION TAGS
The HA epitope tag was first established in 1988 by J. Field and his colleagues, making it one of the oldest protein tags still in use. Its characteristic amino acid sequence YPYDVPDYA is based on a small segment of the viral protein hemagglutinin (abbreviated as HA), one of three integral membrane proteins in the Influenza A virus. The epitope segment was chosen due to its high immunogenicity and antibody affinity which is further increased by fusing multiple epitope copies in a row (three copies = 3xHA). A vast variety of monoclonal primary antibodies from different species have been established against the HA epitope, eliminating the need for expensive protein-specific antibodies and facilitating target protein detection and purification.
Myc
size | 2.5 kDa
available in cloning position B3
The Myc-tag, along with 3xHA and 3xFLAG-tag, is yet another epitope tag designed for protein purification and detection with the help of specific antibodies. It consists of a small peptide whose amino acid sequence (EQKLISEEDL) was originally derived from the transcription factor MYC, which is encoded by the human protooncogene c-myc and plays an important role in the regulation of endogenous gene expression. A pathological, constitutive expression of c-myc may lead to an increased gene expression of genes involved in cell cycle regulation and cell proliferation, thus facilitating the formation of cancer. The gene’s and with it the tag’s name stems from the cancerous disease myelocytomatosis which is observed in birds and is caused by a virus-induced misregulation of c-myc gene expression. When it comes to experimental uses, the Myc-tag can be used for all detection and purification purposes that involve binding of the target protein to antibodies, such as immunoprecipitation or affinity chromatography, and eliminates the need for protein-specific antibodies. This basic part is fused to an HRV 3C protease recognition site.
3xFLAG®
size | 4.2 kDa
available in cloning position B3
Much like the 3xHA tag, the 3xFLAG®-tag is a small polypeptide epitope tag. Yet, unlike the 3xHA-tag, its amino acid sequence is not derived from a naturally occurring protein, but was instead artificially designed to be an epitope for specifically developed primary antibodies. It was first described in 1988 by Hopp et al. as a single Flag tag consisting of the eight amino acid sequence DYKDDDDK. The high content of polyvalently anionic amino acids (like aspartic acid and tyrosine) makes it less likely to interfere with target protein activity. Over the years, many variations of the original Flag-tag were established, including the very commonly used 3xFLAG®-tag patented by SigmaAldrich with the slightly modified sequence DYKDHDG-DYKDHDI-DYKDDDDK. It contains multiple epitope copies, hereby further increasing the tag’s affinity to the anti-Flag antibodies. The 3xFLAG®-tag can be used for all detection and purification purposes that involve binding of the target protein to antibodies, such as immunoprecipitation or affinity chromatography, and eliminates the need for protein-specific antibodies. This basic part is fused to an HRV 3C protease recognition site.
SECRETION TAG
sAP
size | 2.3 kDa
available in cloning position B2
The sAP tag is a secretion signal peptide derived from the secretory protein secreted acid phosphatase 1 (sAP1) which was originally purified from Leishmania mexicana, a human pathogenic Leishmania strain. The secretion of acid phosphatases makes the parasites more resistant to oxidative stress induced by the host’s immune cells. The secretion signal peptide found in sAP1 mediates the exocytosis of the protein after its biosynthesis in the endoplasmatic reticulum (ER). This is extremely useful for recombinant protein production, since target proteins can be harvested directly from cell culture supernatant without the need for cell lysis. Since posttranslational glycosylation of proteins happens within ER and Golgi apparatus along the secretory pathway, the sAP1 signal peptide tag furthermore enables the production of glycosylated proteins in Leishmania. This makes the sAP1 tag a vital part of our cloning kit, since employing Leishmania as an alternative expression host for biopharmaceuticals relies on correct reconstruction of the proteins‘ glycosylation patterns.
OUR PROTEIN OF INTEREST
The COVID-19 pandemic has been sweeping the world for almost two years now, costing millions of lives and causing immense economical, cultural and social damage. The virus responsible for the outbreak of the eponymous respiratory disease was named severe acute respiratory syndrome coronavirus 2 or short SARS-CoV-2. Much like other coronaviruses, its infectiousness relies on multimeric protein structures that project outwards from the virus envelope’s outer surface. Due to their appearance, these protein complexes are often called spikes. They consist of a trimeric complex of the so called spike protein and coordinate the recognition, binding and entry of the virus into the host’s cells. The spike protein itself is a glycoprotein consisting of two separate regions, S1 and S2, of which S1 contains the receptor binding domain (RBD) and mediates cell binding, while S2 contains a fusion peptide and initiates the actual endocytosis of the virus particle. As the entry point into host cells, Sars-CoV-2 makes use of the angiotensin-converting enzyme 2 (ACE2), a transmembrane protein mainly present in intestinal, kidney and cardiac cells and involved in blood pressure regulation. The highly specific binding of the Sars-CoV-2 receptor binding domain to ACE2 is significantly dependent on the posttranslational glycosylations that the RBD undergoes. This makes the recombinant production of correctly glycosylated spike protein very valuable for infectiological research and vaccine development. Thus, by including the Sars-CoV-2 RBD into our cloning kit, we not only demonstrate the significance of our expression system to the advancement in current coronavirus research, we also reveal the glycosylation capabilities of our expression host Leishmania tarentolae.
PROTEASES
TEV Protease
recognition motif | *NLYFQ ▼S, * = E,G,A,M,C,H
fused to B5 GST
The TEV protease is a cysteine protease domain originally derived from the tobacco etch virus (TEV), a plant virus that infects a wide variety of nightshades and weeds, including the eponymous tobacco plant. The protease is known for its extremely high sequence specificity, meaning that it only cleaves proteins upon recognition of a fixed set of amino acid sequences, with little to no off-target effects. This makes it a valuable tool in modern biotechnological research, since it allows for precise protein cleavage at easily predefined positions. By including a TEV recognition motif on either side of a tag sequence, the respective tag can be cleaved off after protein purification via the addition of TEV protease to the mix. Many commercially available TEV proteases are furthermore fused to an affinity tag themselves, making the retrieval of cleaved target protein even more feasible and minimizing the risk of uncoordinated proteolytic activity due to overexposure. The only requirement for a target protein to be fused with our B5 GST-tag and purified using TEV protease is that the protein sequence ought to be free of any intrinsic TEV recognition sequences. This should be ensured beforehand, via sequence analysis or PCR, and any observed recognition sequences should be removed, i.e. by the introduction of silent single point mutations.
HRV 3C Protease
recognition motif | LEVLFQ ▼GP
fused to all B3 parts
HRV 3C is short for human rhinovirus 3C protease (HRV 3C), a cysteine protease that usually helps rhinoviruses to infect human cells by disrupting the cells‘ endogenous transcription activity. By including a HRV 3C recognition motif on either side of a tag sequence, the respective tag can be cleaved off after protein purification via the addition of the protease to the mix. Most commercially available HRV 3C proteases are furthermore fused to an affinity tag themselves in order to facilitate the retrieval of cleaved target protein. The only requirement for a target protein to be fused with an HRV 3C tag and purified using HRV 3C protease is that the protein sequence ought to be free of any intrinsic HRV 3C recognition sequences. This should be ensured beforehand, via sequence analysis or PCR, and any observed recognition sequences should be removed, i.e. by the introduction of silent single point mutations.