DYKDDDDK-peptide is a 8 aa length peptide used for the competitive elution of FLAG fusion proteins (with excess of free DYKDDDDK-peptide) under non-denaturing conditions.
Involvement in COVID-19 disease
Some recombinant proteins, such as Spike glycoprotein and ACE2 are frequently produced using the DYKDDDDK-tag system. In order to purify or detect that proteins, sb-PEPTIDE provides DYKDDDDK-peptide and 3x DYKDDDDK–peptide.
To help your research for COVID-19 disease, we can also provide you with premium quality peptide libraries containing SARS-CoV-2 proteins. Our expertise make us able to synthesize glycopeptides and phosphopeptides to be close from the real content of these peptides. For more information, please visit this link.
A fusion tag, called FLAG and consisting of eight amino acids (AspTyrLysAspAspAspAspLys) including an enterokinase-cleavage site, was specifically designed for immunoaffinity chromatography. It allows elution under non-denaturing conditions [Bio/Technology, 6 (1988) 1204]. Several antibodies against this peptide have been developed. One antibody, denoted as M1, binds the peptide in the presence of bivalent metal cations, preferably Ca(+). Elution is effected by chelating agents. Another strategy is competitive elution with excess of free FLAG peptide. Antibodies M2 and M5 are applied in this procedure. Examples demonstrating the versatility, practicability and limitations of this technology are given.
2- Dubeykovskaya, Z.A et al. Cancer Gene Ther (2019)
TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin β subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.
3- Elena Krachmarova. BioMed Research International (2017)
In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in E. coli cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγ receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.
The FLAG peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags. Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1, M2 and M5. Among the newly identified tag sequences are those that bind only one of the anti-FLAG mAbs and those that bind only two or all three of the anti-FLAG mAbs. Examples of new tag sequences are MDFKDDDDK (which binds mAb M5 and does not bind mAbs M1 and M2) and MDYKAFDNL (which binds mAb M2 and does not bind mAbs M1 and M5). The sensitivity in direct ELISA of some variants was increased, e.g. using mAb M2 it was found that replacing DDDDK in MDYKDDDDK by AFDNL increased the sensitivity in ELISA at least 10-fold. The activity of this peptide was studied in more detail. In different direct ELISAs, in which MDYKAFDNL was synthesized on polyethylene pins, coated onto polystyrene microtiter plates or onto nitrocellulose paper, the activity of this peptide was similar, i.e. increased at least 10-fold over that of MDYKDDDDK. Remarkably, in competitive ELISA the binding activity of soluble MDYKAFDNL was decreased 10-fold over those of soluble MDYKDDDDK or DYKDDDDK. The results seem to suggest that, in solution, the conformation of MDYKAFDNL is more ‘unstructured’ compared to its conformation when coated or linked to a carrier. We postulate that the newly described tag sequences may be used as affinity tags to separately detect, quantify and purify multiple co-expressed proteins and/or subunits.
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