A6002-25mg | FLAG tag Peptide [98849-88-8] Clinisciences

A fusion tag called DYKDDDDK and consisting of eight amino acids Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys including an enterokinase-cleavage site, was specifically designed for immunoaffinity chromatography. It allows elution under non-denaturing conditions. 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 DYKDDDDK peptide. Antibodies M2 and M5 are applied in this procedure1.

The added marker segment is that it should not interfere with the native folding of proteins to which it is attached. Secondly, the marker peptide sequence should be water-soluble and should retain a high degree of exposure on the surface of the protein, so that it can readily interact with its ligand. It should also be suitable for a mild and inexpensive affinity purification procedure. Finally, an easy removal of the marker peptide leading to a native product is also advantageous. Due to this small size, the marker peptide can be encoded by a single synthetic oligonucleotide. It is known that aromatic amino acids are the major factors in antigen–antibody interactions.

Lys at position 3 in the marker sequence leads to a hexapeptide sequence LysAspAspAspAspLys, which ensures a maximum value on the hydrophilicity scale according to Hopp and Woods2 . Such hydrophilic sequences have been shown to express strong antigenicity and are thus likely to adopt a highly exposed conformation in the three-dimensional folding of proteins3. Another virtue of DYKDDDDK is that the longest trypsinogen prosequences are of this length. This enables the removal of the tag and the production of an authentic N-terminus of the fusion protein partner by enterokinase treatment. The DYKDDDDK peptide can be fused to either the N- or C-terminus of a given fusion protein. Nevertheless, the N-terminal fusion has several advantages. Inhibition ELISA experiments showed that the anti-DYKDDDDK antibody M1 binds three to four orders of magnitude better under conditions where the a-amino group of the first amino acid is freely accessible4.

The DYKDDDDK marker peptide fusion system comprises a unique and widely useful technique for protein identification and purification. Elution of the fusion protein can be accomplished either by antibody-mediated affinity chromatography in a calcium-dependent manner, by lowering the pH, or by competitive elution with synthetic peptides1. Although highly selective, the binding capacities are low, making scale-up a costly undertaking. In addition to cost and low capacity, large-scale immunoaffinity chromatography, applied to the production of therapeutic proteins has several disadvantages: ligand leakage, instability, and need for validation of antibody production. The stability of the affinity chromatography column depends on the nature and source of the crude extracts. Furthermore, the DYKDDDDK tag, designed to be immunogenic, must be removed from therapeutic proteins. In most cases, this can be accomplished with enterokinase. However, contaminating proteases may also produce undesired cleavages. Despite these drawbacks, the DYKDDDDK fusion is useful in research and development: DYKDDDDK proteins can be readily purified and assayed by ELISA or any other immunochemical detection method, thus expediting the raising of antisera against a desired protein and characterization studies.

References:
1. A. Einhauer, A. Jungbauer. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J. Biochem. Biophys. Methods 49 2001 455–465.
2. Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A 1981;78:3824–8.
3. Hopp TP. Protein surface analysis: methods for identifying antigenic determinants and other interaction sites. J Immunol Methods 1986;88:1–18.
4. Power BE, Ivancic N, Harley VR, Webster RG, Kortt AA, Irving RA, et al. High-level temperature-induced synthesis of an a

1. Takushi Miyoshi, Qianli Zhang, et al. "Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures." Cell Rep. 2021 Feb 2;34(5):108708. PMID: 33535030
2. Denghui Wei, Weixiang Zhan, et al. "RAB31 marks and controls an ESCRT-independent exosome pathway." Cell Res. 2021 Feb;31(2):157-177. PMID: 32958903
3. Ji R, Xu X, et al. "Regulation of adiponectin on lipid metabolism in large yellow croaker (Larimichthys crocea)." Biochim Biophys Acta Mol Cell Biol Lipids. 2020;1865(8):158711. PMID: 32289502
4. Ilaria Ceppi, Sean M. Howard, et al. "CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection." bioRxiv. 2019 October 01.
5. Marcum RD, Radhakrishnan I. "Inositol phosphates and core subunits of the Sin3L/Rpd3L histone deacetylase (HDAC) complex up-regulate deacetylase activity." J Biol Chem. 2019 Jul 29. pii: jbc.RA119.009780. PMID: 31358618
6. Ter Beek J, Parkash V, et al. "Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase." Nucleic Acids Res. 2019 Jun 20;47(11):5712-5722. PMID: 30968138
7. Wang M, Hu J, et al. "High glucose-induced ubiquitination of G6PD leads to the injury of podocytes." FASEB J. 2019 Feb 20:fj201801921R. PMID: 30785802
8. Zi-Wei Chen, John R Bracamontes, et al. "Multiple Functional Neurosteroid Binding Sites on GABAA Receptors." bioRxiv. 2018 June 29.
9. Heredia JD, Park J, et al. "Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning." J Immunol. 2018 Apr 20. pii: ji1800343. PMID: 29678950
10. Sarah Zernia, Ronny Frank, et al. "Surface‐Binding Peptide Facilitates Electricity‐Driven NADPH‐Free Cytochrome P450 Catalysis." ChemCatChem. 2017 November19.
11. Banks CJ, Rodriguez NW, et al. "Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-mediated Inhibition of Mitochondrial Respiration." Mol Cell Biol. 2017 Jul 24. pii: MCB.00354-17. PMID: 28739857
12. Lopez J, Bessou M, et al. "Mito-priming as a method to engineer Bcl-2 addiction." Nat Commun. 2016 Feb 2;7:10538. PMID: 26833356

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Physical Appearance	A solid
Storage	Desiccate at -20°C
M.Wt	1012.97
Cas No.	98849-88-8
Formula	C₄₁H₆₀N₁₀O₂₀
Synonyms	H-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-OH
Solubility	≥50.65 mg/mL in DMSO; ≥210.6 mg/mL in H2O; ≥34.03 mg/mL in EtOH
Chemical Name	DYKDDDDK Peptide
SDF	Download SDF
Canonical SMILES	C1=CC(=CC=C1CC(C(=O)NC(CCCCN)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CC(=O)O)C(=O)NC(CCCCN)C(=O)O)NC(=O)C(CC(=O)O)N)O
Shipping Condition	Small Molecules with Blue Ice, Modified Nucleotides with Dry Ice.
General tips	We do not recommend long-term storage for the solution, please use it up soon.

Description	DYKDDDDK tag Peptide is a 8 amino acid peptide with an enterokinase-cleavage site used for the purification of recombinant proteins.
Targets	anti-DYKDDDDK M2 antibody
IC50

Purity = 99.90%

FLAG tag Peptide [98849-88-8]

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