Cloning and gene comparison of the cDNA encoding the acidic proteinase After obtaining the partial DNA sequence of MCAP, specific primers were designed for the amplification of 3′-RACE and 5′-RACE of aspartic proteinase gene from the first-strand cDNA of M. circinelloides by SMART™
RACE PCR. Bafilomycin A1 The full-length cDNA of the aspartic proteinase from M. circinelloides was amplified from the 5′ first-strand, while the full-length MCAP encoding the aspartic proteinase was amplified from genomic DNA of M. circinelloides. By comparing the nucleotide sequence of aspartic proteinase amplified from the 5′first-strand cDNA with the sequence amplified from the genomic DNA of M. circinelloides, we found
that the whole MCAP has an intron of 63 bp long and encodes 394 amino acid residues (Figure 2). The amino acid sequence of M. circinelloides MCAP was further aligned with the M. bacilliformis sequence and with non-redundant protein database using BLASTX 2.2.24. The highest similarity between the MCAP amino acid sequence and a M. bacilliformis homolog was found to be 88% identity. The identity with R. oryzae (accession number ACL68088), R. microsporus (accession number CAA72511), R. microsporus var. chinensis (accession number AAB59305), R. niveus (accession number CAA40284), and S. racemosum (accession number AAC69517) was 66, 65, 64, 63, and 59%, respectively. Figure 2 The Nucleotide and deduced amino acid sequence of MCAP protein.
CDK inhibitor The deduced amino acid sequence is shown under the nucleotide sequence. The arrow indicates the proposed signal peptide cleavage site and lowercase letters indicate nucleotides in the intron sequence. The proposed catalytic Asp residues (motifs DTGS and DTGT) are boxed. The potential N-glycosylation site is underlined. Asterisk indicates the position of the stop codon (TAA). Signal peptide sequence and N-glycosylation site The analysis of the amino acid sequence by a SignalP 3.0 server identified a cleavage signal sequence Axenfeld syndrome site between positions Ala21 and Ala22 in the MCAP protein (http://www.cbs.dtu.dk/services/SignalP/). The putative signal peptide corresponding to the first 21 amino acids; MKFSLVSSCVALVVMTLAVDA, shows features of signal peptides, such as a highly hydrophobic region. Additionally, by using the NetNGlyc v1.0 server (Center for Biological Sequence Analysis, Technical University of Denmark DTU), one potential N-glycosylation site; Asn–X–Ser/Thr, was found to be at positions Asn331 in the MCAP (Figure 2). Protein expression, purification and SDS-PAGE analysis To analyze the expression of MCAP gene in P. pastoris, a set of recombinant plasmids carrying either the partial or the whole MCAP gene, was cloned into the P. www.selleckchem.com/products/fosbretabulin-disodium-combretastatin-a-4-phosphate-disodium-ca4p-disodium.html pastoris expression vector pGAPZα-A. The secreted MCAP forms were separated by SDS-PAGE.