An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments.
The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity. Using the calcium-dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD). Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods. Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp. The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E. coli is processed correctly. Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position. Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins.
Knappik
A
A
Department of Biochemistry Universität Zürich, Switzerland.
Plückthun
A
A
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid; D007128:Immunoglobulin Fragments / Q000032:analysis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology*; D011994:Recombinant Proteins / Q000032:analysis* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D018698:Glutamic Acid; C085706:FLAG peptide