1 The methods described above have been used successfully for a few streptavidm-containing chimera constructs (13,14) However, the conditions may need to be adjusted or optimized for a particular streptavidin-partner protein chimera to maximize the production and final yield of functional chimera molecules
2. Rich media, such as LB medium, can also be used for expression of a streptavidin-partner protein chimera and could give higher expression efficiency than with M9 minimal medium In our experience, however, the use of rich media often causes cellular degradation of the partner protein moiety of an expressed chimera, thereby reducing the final yield The streptavidin moiety is generally resistant to cellular proteinases, even in a denatured form Thus, rich media should be used for expression if the stability of a partner protein against cellular proteolysis is sufficiently high
3 During punfication, each fraction should be analyzed by SDS-PAGE to find the optimal purification procedure for a particular streptavidin-partner protein chimera.
4 If degradation of chimera molecules during purification is observed, proteinase inhibitors, such as phenylmethylsulfonyl fluoride, pepstatm, leupeptin, and E-64, are added to all solutions used for purification. Because the streptavidin moiety is extremely stable against proteolysis when it is folded, the degradation of the chimera is likely to occur within the partner protein moiety. Such degradation of the chimera can be analyzed by Western blot analysis using anti-streptavidin after SDS-PAGE. Degradation products of the expressed chimera are detected by labeling the streptavidin moiety with anti-streptavidin. The determination of the size of each labeled protein component allows one to locate the sites in the partner protein moiety where proteolysis occurred
5. The biotm-binding ability of purified chimera can be determined by a gel filtration method (15) using a desalting column, such as PD-10 columns (Pharmacia, Piscataway, NJ), and radiolabeled biotin, such as D-[carbonyl-XAC]b\otm (Amersham, Arlington Heights, IL) and d-[8,9-3H]biotm (Amersham and DuPont NEN, Boston, MA). Alternatively, the biotin-binding ability can also be determined semiquantitatively using nonradioactive methods, such as enzyme-binding assays (16). Usually, the purified chimera has almost full biotin-bmding ability, as it has been purified by using its biotm-binding ability
6 The purified chimera is generally tetrameric, the subunit association of the chimera is determined by the streptavidin moiety whose subunit association is exceptionally stable However, the subunit association of a particular streptavidin-partner protein chimera should be determined experimentally by using, for example, gel filtration chromatography.
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