| 摘要 |
The present invention relates to a method to detect the interaction between a target ligand and a moiety of interest using an adenylate cyclase enzyme (AC) and calmodulin (CaM) as interacting partners, said method comprising:
i) expressing in a suitable host
(a) a low number of molecules of a first chimeric polypeptide containing AC, and
(b) a low number of molecules of a second chimeric polypeptide containing CaM,
wherein said AC in said first chimeric polypeptide and/or said CaM in said second chimeric polypeptide has decreased affinity for its interacting partner,
wherein said AC in said first chimeric polypeptide is fused to a moiety of interest and said CaM in said second chimeric polypeptide is fused to a target ligand, or conversely,
and wherein, when said moiety of interest and said target ligand interact, said AC is activated, and
ii) detecting the activation of said AC. The present inventors herein show that only one AC/CaM complex per cell is sufficient to confer a selectable trait to the host cell. Unexpectedly, even less than one AC/CaM complex per cell can be sufficient to confer a selectable trait to the host cell. This surprising result confers a very high sensitivity, that is helpful for screening high affinity interactions, such as antigen-antibody interactions. Moreover, the low expression of the chimeric proteins that is achieved in the present invention allows to characterize toxic moieties, what was not possible before. |
