Flag Tag and Flag Tag Antibody for Protein Purification

Affinity labels refer to proteins or short peptides that have high specificity for specific biological or chemical ligands, and have the advantages of high affinity, mild purification conditions, simple purification procedures, and wide applicability. The Flag fusion tag, as one of the best, is a short hydrophilic peptide specially designed for protein purification and detection, consisting of eight amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). The Flag tag can be located on the C or N terminal of the protein, and the system has been widely used in a variety of cell types, including bacteria, yeast, and mammalian cells. The fusion protein can be detected and purified by the corresponding Flag labeled antibody, and the purification efficiency of the target protein can be greatly improved.

1. Function of Flag Labels

As an artificially designed fusion tag, these eight amino acid residues are not randomly arranged, but are closely related to the action of the Flag antigen. The second amino acid in the Flag sequence is tyrosine (Tyr), which belongs to the aromatic amino acid and is the main factor of antigen-antibody specific reaction. The negatively charged aspartic acid (Asp) at the N terminal can assist the antigenicity of Tyr, because the possibility of aromatic amino acids acting in high polarity environment is higher than that in low polarity environment. The six amino acids near the C-terminal (Lys-Asp-Asp-Asp-Asp-Lys) constitute a hydrophilic sequence, which may form a highly exposed three-position protein configuration, which theoretically enables the sequence to achieve maximum hydrophilicity and greatly enhances the antigenicity of the Flag fusion tag.

2. Advantages of Flag Labels

Flag, as a tag protein, has the following advantages after fusion to express the target protein:

(1) The sequence is short and can be encoded by only one synthetic oligonucleotide chain;

(2) Under crystallization conditions, the conformation of the Flag fusion protein is almost identical to that of the pure target protein, and it usually does not interact with the target protein and usually does not affect the properties and functions of the target protein, so there are researchers to conduct downstream studies on the fusion protein;

(3) The target protein with Flag label can be fused directly through the Flag affinity chromatography, which is non-denatured purification and can purify the active fusion protein with high purification efficiency;

(4) Anti-FLAG antibodies can effectively recognize the Flag label, thus facilitating the detection and identification of fusion proteins containing Flag by Western Blot, ELISA and other methods;

(5) The Flag fusion tag contains a cutting site of enterokinase, which can recognize the five amino acids of the C-terminal of the short peptide (Asp-Asp-Asp-Asp-Lys), and natural non-fusion proteins can be obtained after the tag is removed by enterokinase treatment.

3. Removing the Flag Label

At present, there are two main methods to remove affinity labels: one is to use specific proteases to remove fusion labels; The second is the introduction of protein intrapeptides (or intrapepons) into the fusion protein. Specific protease cleavage sites are introduced: The proteases enterokinase, thrombin, Factor Xa, tobacco scratch virus (TEV), genenaseⅠ and 3C have cleavage sites between the fused short peptide and the target protein.

(1) Intestinal kinase: Intestinal kinase is a protease often used in the removal of N-terminal labels (especially the Flag label fused in the protein N-terminal), which can accurately identify the sequence composed of the five amino acid residues of D-D-D-D-K, and the removal efficiency mainly depends on the first amino acid after lysine (K). The Flag label (DYKDDDDK) contains an enterokinase cutting site, and after digestion, a Flag affinity label containing only 4 amino acid residues can be obtained. The cDNA sequence encoding the catalytic site of bovine enterokinase has been cloned and expressed in mammalian cells and E. coli. Enterokinase has the characteristics of high activity, wide pH range, and can be cut at a variety of denaturants and detergent concentrations, and the cut product does not contain excess amino acids. Hosfield et al. used the method of ligation independent cloning (LIC) to introduce an amino acid residue between the Flag tag and the target gene to form a -DYKDDDDK-X-R sequence (X represents the introduced residue and R represents the target gene). The calmodulin gene was introduced into the above sequence as R to study the effect of residue X on the cutting efficiency of enterokinase. The experimental results showed that the amino acid side chain near the cutting site was longer and the volume was smaller, so the cutting was more effective. When X is Lys, Ala, Leu, Ile, Phe, Glu, Met, Asp and Asn, it has a better cutting efficiency, which can reach more than 80%. However, when X is Tyr or Pro, the cutting efficiency drops below 70% because of the increased steric hindrance. The above statement is very important for obtaining natural proteins. Therefore, the Flag tag can be directly connected to the target gene sequence by LIC method, and after expression, the product is purified by Flag tag, and then the natural protein is obtained by cutting and removing the Flag tag by enterokinase, and its properties and functions are studied.

(2) Thrombin: Also a very widely used protease, the main feature is that the recombinant protein after thrombin cleavage will retain two amino acid residues at the C end of the cleavage site. Thrombin can recognize two types of amino acid sequences, one is X4-X3-P-R[K] -X1-x2, the other is X2-R[K]-X1, and the recognition effect of thrombin on the former sequence is relatively ideal. The experiment showed that if 5 amino acid residues were designed between the cutting site and the N-terminal label, the cutting activity of thrombin could be increased.

(3) Factor Xa: Like the above proteases, factor Xa is also a more efficient tool enzyme for removing fusion tags, which can specifically recognize the I-E[D]-G-R-X1 sequence and cut the fusion tag from its C-terminal. This recognition is unique among many proteases, but the cutting of factor Xa requires a long incubation time and is not efficient.

Overview and Application of Anti-FLAG Monoclonal Antibody

1. Research progress of anti-FLAG monoclonal antibody

At present, a total of three monoclonal antibodies against the Flag label have been developed and put into use, namely M1, M2 and M5. The three antibodies differ in their use, especially when they bind to different antigens. M1 monoclonal antibody is the first to be applied, and its characteristic is that it can only bind to the Flag antigen with the participation of Ca2+, and it is often used in the identification and purification of many recombinant fusion proteins with N-terminal Flag labels. Amino acid scanning experiments show that the binding of M1 monoclonal antibody is mainly the first four amino acids and requires a free N-terminal amino group. That is, if the Flag tag is fused to the C-terminal of the protein, or if there are other amino acid residues in the N-terminal of the fused protein with the Flag tag, the M1 antibody cannot function. In order to solve the above situation, anti-FLAG M2 monoclonal antibodies subsequently appeared, which greatly expanded the field of application of Flag tags. Since the binding of the M2 antibody to the antigen does not depend on Ca2+, the chelating agent (such as EDTA) cannot act on the antigen-antibody complex on the adsorption column. Another important advantage of the M2 antibody is that it allows for the presence of excess amino acid residues before the Flag sequence, which can be applied to N-terminal Met-Flag and C-terminal Flag fusion proteins. Similar to the generalized M2 monoclonal antibody, the binding of the third monoclonal antibody M5 is also independent of Ca2+ and is mostly used for sequences similar to N-Met-Flag-C, which has a very high affinity for this fusion protein. Therefore, M5 monoclonal antibody is the antibody of choice for detecting the Flag fusion protein expressed in the cytoplasm. M2 monoclonal antibody can bind to all types of N-terminal Flag fusion proteins mentioned above, so it has versatility.

2. Production and identification of anti-FLAG monoclonal antibody

(1) Production and identification of complete antigens of Flag label: The Flag label is coupled with carrier proteins by carbonized imide method. Carrier proteins include bovine serum albumin (BSA), ovagmin (OVA) and keyhole hemocyanin (KLH), and three immune antigens are prepared. The antigens synthesized by the three carrier proteins are mutually coated antigens.

(2) Production of hybridoma cell line with Flag monoclonal antibody: the antigens and adjuvants mentioned above were used for animal immunization, and the titer was subsequently detected by indirect ELISA method. The hybridoma cell lines with the best titer were selected for fusion, and the positive clones were screened and amplified, and finally hybridoma cell lines were obtained.

(3) Production and identification of monoclonal antibody with Flag label: ascites was produced by Golding method, and purified by n-caprylic acid-ammonium sulfate method, and finally purified ascites was obtained. The titer, purity, monoclonal antibody subtype and subclass, cross-reaction and relative affinity of ascites were determined to complete the Production and identification of Flag monoclonal antibody.

3. Application of anti-Flag monoclonal antibody

(1) For the detection and purification of fusion proteins: although many non-affinity tags have biological activity and can be used for the detection of fusion proteins, these non-affinity tags may affect some of their biological activities due to the limitations of the expression environment or detection conditions. Therefore, antibodies are still the most important method for analyzing fusion protein expression. With the participation of anti-fusion label monoclonal antibodies, the protein levels of the expressed products can be easily detected by enzyme-linked immunosorbent assay (ELISA) and western blot. This is more significant for the expression of unknown proteins. In addition to the detection of fusion proteins, another major use of anti-fusion tag antibodies is the purification of corresponding fusion proteins. Studies have shown that the purification efficiency of immunoaffinity chromatography using anti-fusion label antibodies is very high, and only one step can obtain 103 ~ 104 times the purification effect. However, it must be based on the specific binding of the antibody to the target protein. In the purification process, due to the specific binding site between the monoclonal antibody and the corresponding antigen, only a single type of interaction can be destroyed during elution to release the target protein. This allows greater protection of the target protein, and the monoclonal antibodies used for purification can be reused repeatedly.

(2) For exploring the structure and function of proteins: people insert tags in different parts of the target protein molecule, and anti-label antibodies can be used to study its molecular structure. Anti-label antibodies can also be used to study the function of protein molecules and specifically the function of membrane receptors. After the binding of different ligands to the membrane receptor, the aggregation of the receptor is generally first caused by cross-linking, that is, the receptor dimerizes or polymerizes, and then the polymerized receptor transmits signals to the cell. This aggregation is often necessary for the receptor to initiate signal transduction, and may be sufficient for some receptors. Therefore, in experiments, we can use the cross-linked activated receptor of anti-label protein antibodies to replace the natural ligand to study the signal transduction or function of the receptor. In addition, inserting tags into specific domains of signal transduction molecules can also be used to study the signal transduction pathways of molecules.

(3) Others: Anti-label antibodies can also act as anchor proteins to fix tagged fusion proteins to the carrier. Compared with other solidization methods, antibody immobilization can maintain the biological activity and stability of fusion protein, so it has a broad application prospect in enzymology and drug screening.

(4) Used to find interacting proteins: Studies have shown that most affinity tags do not affect the spatial conformation and biological function of the target protein when applied. Thus, anti-labeling antibodies can be used after fluorescent labeling to find proteins that interact with the fusion protein (including ligands or receptors of the target protein).

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