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背景说明：FLAG peptide是一种八个氨基酸组成的多肽 (Asp-Ty-rLys-Asp-Asp-Asp-Asp-Lys)，具有肠激酶切割位点；FLAG peptide用于抗体介导的重组蛋白的识别和纯化。
FLAG peptide is an eight amino acids peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) with an enterokinase-cleavage site; designed for antibody-mediated identification and purification of recombinant proteins.
In Vitro：Fusion protein technology has become an important tool for solving numerous problems linked to recombinant protein production. The properties of the additional tag facilitate identification and provide a one-step purification procedure of the fusion protein by passing cell extracts or supernatants through columns of an appropriate matrix. FLAG peptide allows elution under non-denaturing conditions. Several antibodies against FLAG peptide have been developed. One antibody, M1, binds the peptide in the presence of bivalent metal cations, preferably Ca2+. Elution is effected by chelating agents. Another strategy is competitive elution with excess of free FLAGe peptide. Antibodies M2 and M5 are applied in this procedure.
The Flag-tag is first described as a calcium-dependent epitope of a monoclonal antibody. It is a highly acidic octapeptide which can be N-terminally fused to the protein of interest. As a very hydrophilic peptide the Flag–tag has a high surface probability. Flag-fusion proteins can be captured by an immunoaffinity column in the presence of Ca2+ and eluted byEDTA at low concentrations, neutral pH and thus, nearly physiological conditions.
参考文献：[1] Miyoshi T , Zhang Q , Miyake T ,et al.Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures[J].Cell Reports, 2021, 34(5):108708.DOI:10.1016/j.celrep.2021.108708.;
[2] A,Einhauer,and,et al.The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins[J].Journal of Biochemical and Biophysical Methods, 2001.DOI:10.1016/S0165-022X(01)00213-5.
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