Fig. 6.

Rationale of designing a SNAP-23 transmembrane fusion protein SNAP-23 Cys⁻-MDR3_1–145, Multi-pass transmembrane proteins expressed in human or rat cells were searched in UniprotKB database. Out of around 1000 hits obtained, three proteins namely MDR3, NaKATPase alpha-2 subunit and CLP24 were narrowed down after extensive database mining. Based on the database information, these proteins localize to the plasma membrane, have known sequence, exhibit constitutive expression, and would not likely affect the function of SNAP-23, and were thus suitable for cloning into SNAP-23. Other proteins showing tissue specific or organelle specific expression; or with unknown sequence; or signaling functions were excluded. Further for all individual transmembrane domains from these three proteins, topology prediction was done using TMMOD model [26], which led to narrowing down of five suitable transmembrane domains for cloning (MDR3_1–145, MDR3_41–145, NaKATPase alpha-2 subunit_285–340, NaKATPase alpha-2 subunit_940–1006 and CLP24_134–195). Shown here is a representative TMMOD output of MDR3_1–145 domain with two transmembrane regions; and correct orientation of N and C terminals facing inside the cytosol. MDR3 protein is constitutively expressed in various human cancer cell lines [27] and topology mapping of individual transmembrane domains of MDR3 has been done in great detail [28,29]. Based on these studies we chose MDR3_1–145 transmembrane domain for cloning. TMMOD model was first used to predict the transmembrane topology of fusion protein SNAP-23 Cys⁻-MDR3_1–145. Shown here is the topology prediction showing two transmembrane regions and the correct expected orientation of the fusion protein with N and C terminals of SNAP-23 facing inside the cytosol. Fusion construct SNAP-23 Cys⁻-MDR3_1–145 was then made by cloning MDR3_1–145 domain into the linker region of SNAP-23 Cys⁻ mutant using a unique restriction enzyme site Ahd I.