Identification of new claudin family members by a novel PSI-BLAST based approach with enhanced specificity.
In an attempt to develop a novel strategy for the identification of new members of protein families by in silico approaches, we have developed a semi-automated procedure of consecutive PSI-BLAST (Position-Specific-Iterated Basic Local Alignment Search Tool) searches incorporating identificiation as well as subsequent validation of putative candidates. For a proof of concept study we chose the search for novel members of the claudin family. The initial step was an iterated PSI-BLAST search starting with the PMP22_Claudin domain of each known member of the claudin family against the human part of the RefSeq Database. Putative new claudin domains derived from the converged list were evaluated by a validating PSI-BLAST in which each sequence was assessed for finding back the starting set of known claudin domains. The local PSI-BLAST searches and validation were automated by a set of PERL scripts. With this strategy a total of three additional putative claudin domains in three different proteins were identified. One of them was subjected to further characterization and was shown to exhibit claudin-like features in terms of protein structure and expression pattern. The strategy we present is an efficient and versatile tool to identify novel members of domain-sharing protein families. Low rates of false positives achieved by inclusion of a validation step into the in silico procedure make this strategy particularly attractive to select candidates for subsequent labor-intensive wet bench characterization.
Wu
Jun
J
Ganymed-Pharmaceuticlas AG, Freiligrathstrasse 12, 55131 Mainz, Germany.
Helftenbein
Gerd
G
Koslowski
Michael
M
Sahin
Ugur
U
Tureci
Ozlem
O
D000595:Amino Acid Sequence; D019295:Computational Biology; D030562:Databases, Protein; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein
D008565:Membrane Proteins