Screening of phage displayed human liver cDNA library against dexamethasone.
This paper described an attempt to establish a new method to screen the target biomolecule from phage displayed cDNA library against small molecule drug insoluble in water. Dexamethasone was selected as the model drug, and the screening was carried out in an Eppendorf tube packed with the drug. The whole procedure was monitored by PCR with the enriched specific phage clone as the template. After four rounds screening, the PCR products of selected phages with the lengths of 400 and 600 bp were sequenced, and revealed identical sequences with cytochrome c oxidase subunit III and albumin respectively by GenBank searching. Furthermore, frontal analysis-capillary electrophoresis (FA-CE) was performed to study the interaction between dexamethasone and albumin, and the binding constant was calculated to be 1.153 x 10(3), validating the weak specific interaction between the drug and the target protein. All these results demonstrated that with insoluble drug as the solid phase directly, the screening of target large molecule expressed in phage display cDNA library was feasible, which might pave an easy way to screen the candidate drug targets.
Yu
Xiaoming
X
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, PR China.
Zhao
Peng
P
Zhang
Weibing
W
Zhang
Lihua
L
Zhang
Yukui
Y
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002138:Calibration; D018076:DNA, Complementary / Q000235:genetics*; D003907:Dexamethasone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D019075:Electrophoresis, Capillary; D005240:Feasibility Studies; D015723:Gene Library; D005820:Genetic Testing; D006801:Humans; D016014:Linear Models; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012015:Reference Standards; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012709:Serum Albumin / Q000378:metabolism; D012995:Solubility; D013698:Templates, Genetic
D018076:DNA, Complementary; D012709:Serum Albumin; D003907:Dexamethasone