BioTechniques Vol. 7, No. 6 (1989) 580-589. A Calcium-Dependent Antibody for Identification and Purification of Recombinant Proteins Kathryn S. Prickett, David C. Amberg and Thomas P. Hopp Immunex ABSTRACT We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase. INTRODUCTION Modern molecular biology has made it possible to clone and express proteins in a variety of host organisms. However, purification of recombinant proteins is an uncertain and often tedious task, usually requiring multiple steps, each of which is dependent upon the specific characteristics of the protein. In some cases harsh treatments are required during the purification, so that complicated renaturation procedures are necessary before biological activity can be restored. The yield of recombinant proteins can sometimes be increased by expressing them as part of a larger fusion protein. Such products have been made cytoplasmically (6,12) or secreted from yeast (4) or E. coli (19) cells. Although fusion per se seems to increase yield in many cases, it does nothing inherently to improve purification yield. A more sophisticated approach is to fuse the desired protein to a polypeptide sequence that is useful for identifying and purifying the product (1,5). In the past, the latter approach has had several drawbacks. First, the fusion protein products may fail to fold properly into a native, active state (15), and, second, it is often difficult or impossible to remove the additional amino or carboxyl-terminal sequence from the desired protein product. To circumvent some of these problems, we have designed a fusion sequence that allows rapid purification of recombinant proteins under very mild conditions. The fusion sequence is a short hydrophilic peptide, AspTyrLysAspAspAspAspLys, that can be engineered onto the N-terminus of a protein using short oligonucleotides. This marker sequence, or Flag™ segment, can be identified by a monoclonal antibody that can also be used as an immuno-affinity gel reagent to purify the resulting fusion protein. The antibody reacts with specific amino acids of the marker sequence and is dependent on the presence of Ca ++ for binding. This allows a mild, one-step purification of fusion proteins when the affinity gel is eluted by removing or chelating calcium from the washing buffer. The peptide can be subsequently detached from the protein by treatment with the protease enterokinase, which cleaves immediately after the -AspAspAspAspLys- sequence.