Similar protocols

Pipeline publication

[…] gonucleotides flanking the predicted translational start and stop codons (20 nucleotides upstream, 5′-TCGGGACCGACCACGAACG-3′; 63 nucleotides downstream, 5′-CGATTCTGTCTTGAAGCCCGACT-3′). The PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, WI) followed by sequencing with a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The nucleotide sequence reported in this paper has been deposited in the DDBJ database under accession number AB901366., The C. cinerea genome database (http://www.broadinstitute.org/annotation/genome/coprinus_cinereus/MultiHome.html) was searched with the amino acid sequence of CDH from P. chrysosporium (accession no. AAB92262) using the TBLASTN algorithm , . The search was carried out with standard settings and the BLOSUM 62 matrix. The amino acid sequences of candidate genes were scanned for the presence of signal peptides using SignalP version 4.1 software at the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/) and searched against the protein database at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The homology structure model was constructed using Protein Homology/analogy Recognition Engine (Phyre) version 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/) . The CcSDH model was used for preparing a figure in which PQQ of sGDH from Acinetobacter calcoaceticus and heme in the cytochrome domain of P. chrysosporium CDH (PDBID 1CRU and 1D7C, respectively) were superimposed to the model. The structure based alignment was created during the process of homology modeling using the Phyre2 server, and the corresponding figure was prepared using ESPript ver. 2.3 ., The expression vector was constructed as described , . Two different oligonucleotide primers were designed based on the nucleotide sequence of the mature protein to allow for ligation into the […]

Pipeline specifications

Software tools TBLASTN, SignalP, Phyre