要旨
INTRODUCTION
Recombinant antibody fragments are powerful tools for the preparation of membrane protein complexes, yielding highly purified material suitable for crystallization in a single immunoaffinity chromatography step. They have been invaluable for obtaining well-ordered crystals of the cytochrome c oxidase from Paracoccus denitrificans (Ostermeier et al., 1995, 1997) and of the yeast cytochrome be1complex (Hunte et al., 2000; Lange et al., 2001; Lange and Hunte, 2002).
Compared with classical methods like ion-exchange or size-exclusion chromatography, immunoaffinity chromatography offers an excellent purification factor—usually between 100 and 1O,OOO—due to the high specificity of the interaction between antigen and antibody or antibody fragment. However, the strength of this interaction also limits the availability of suitable elution protocols if the antibodies or their fragments are covalently coupled to activated matrices or trapped by immobilized protein A or G. Alternatively, engineered antibody fragments can be noncovalently and reversibly bound to an appropriate matrix via an affinity tag, such as, for example, the Strep-tag (Schmidt and Skerra, 1993). The protein is consequently eluted as a co-complex with the antibody fragment (see Fig. 1) and can be used as such for crystallization trials. This indirect immunoaffinity chromatography approach combines the advantages of antigen/antibody interaction and affinity tag techniques, namely high specificity and well-established purification protocols of broad applicability. Furthermore, the technique allows isolating the protein from its original source, as well as from an expression system in its native form without the attachment of affinity markers. Starting from whole cells, the protein of interest can be obtained in very pure form within about eight hours. Obviously, the purified protein is present as a co-complex with the antibody fragment bound. Thus, the latter should not affect the biological activity of the protein unless this is desired. Cytochrome c oxidase from Paracoccus denitrificans (Kleymann et al., 1995) as well as the quinol oxidases from P. denitrificans and Escherichia coli (Ostermann, 2000) have efficiently been purified by one-step indirect immunoaffinity chromatography using recombinant antibody fragments without affecting the functional properties of these membrane protein complexes.
