Knappik A, Plückthun A
Department of Biochemistry Universität Zürich, Switzerland.
Biotechniques. 1994 Oct;17(4):754-61.
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.
市售的单克隆抗体M1和M2是针对FLAG序列DYKDDDDK产生的，并与其高度特异性结合。利用钙依赖性M1抗体以及连接到在大肠杆菌中表达的抗体McPC603各种片段N端的FLAG标签，我们发现M1抗体与该序列的一个短得多的版本（DYKD）以几乎相同的亲和力结合。由于大多数抗体轻链以天冬氨酸开头，因此在N端仅添加三个额外的氨基酸就足以使用标准免疫方法检测和定量表达的抗体片段。同样，重链可以用序列DYKD特异性检测，由于大多数重链不以天冬氨酸开头，所以这需要四个额外的氨基酸。两条链运输到大肠杆菌周质所必需的信号序列被正确加工。此外，我们研究了FLAG序列第五位氨基酸对M1抗体结合亲和力的影响，发现该位置的谷氨酸使蛋白质印迹中的灵敏度比在该位置含有天冬氨酸残基的原始长FLAG序列提高了六倍。总之，改进后的FLAG是一种用于重组蛋白灵敏检测和一步纯化的通用工具。
