Basic–Liver, Pancreas, and Biliary TractFunctional expression of the canalicular bile salt export pump of human liver*,**
Section snippets
Materials
[3H]Taurocholic acid (2 Ci/mmol) and [3H]cholic acid (27.5 Ci/mmol) were obtained from NEN Life Science Products (Boston, MA). [3H]-labeled glycocholic, taurochenodeoxycholic, and tauroursodeoxycholic acids of high specific activity (14–30 Ci/mmol) were prepared as described previously.14, 15, 16 All other chemicals and reagents were of analytic grade and were readily available from commercial sources.
Isolation of human BSEP
Cloning of human BSEP was started with the amplification of a 450–base pair complementary DNA
Results
Isolation of BSEP from human liver required repeated screening of a human liver cDNA library with successive isolation of several complementary cDNA fragments, which then could be ligated together to yield the full-length human BSEP cDNA. Our experience supports previous experiences that full-length clones of BSEP are absent from human cDNA libraries.12 The reason for the failure to generate full-length BSEP cDNA clones could be explained by the presence of cryptic E. coli promoter sequence
Discussion
The present study shows the isolation and functional characterization of the BSEP of human liver. In contrast to its rat and mouse orthologs, the successful cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif (Figure 1). In addition, its functional expression in Sf9 cells was dependent on the back-mutation of some, most probably artificially introduced, amino acid replacements to the published amino acid sequences (Table 1). Thus, the only “abnormal” amino acid
Acknowledgements
The authors thank Monika Gersbach for excellent technical assistance.
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Address requests for reprints to: Peter J. Meier-Abt, M.D., Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland. e-mail: [email protected]; fax: (41) 1-255-4411.
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Supported by the Swiss National Science Foundation (grant 31-64140.00).