, 2005) and in M. grisea GUY II (Leung et al., 1988). Activity was also detected in commercial enzyme preparations, such as Celluclast®. A three-step protocol was elaborated to purify the enzyme secreted by T. reesei RUT-C30. Using RNAse B as a test
substrate, the yield and specific activity enhancement could be estimated (Table 1 and Fig. 1). Taking advantage of the absence Selumetinib clinical trial of a carbohydrate-binding module in the Endo T, the Avicel adsorption step was efficient in removing the bulk of the cellulases (14-fold enrichment), although a substantial decline (61%) in yield was observed. Anion exchange chromatography yielded a large fraction at 180–300 mM salt, active against yeast invertase as detected by PAGE Gefitinib solubility dmso band shifting. This purification step resulted in a substantial enrichment (172-fold) and almost no loss of activity. A
final 1300-fold purification with a 25% yield of activity and 870 μg of extracellular protein were obtained. Under reducing conditions of the purified protein, SDS-PAGE revealed two closely migrating protein bands in the 30–33 kDa range (Fig. 1). N-terminal sequencing of the minor fraction with the highest apparent molecular weight (Fig. 1, lane 6) indicated a contaminating RNAse from T. reesei. Its presence in the final Endo T preparation was not detrimental to the results of our study. CNBr treatment before trypsin digestion of the major fraction with the lowest molecular weight on gel (Fig. 1, lane 6) yielded a large peptide of 3212 Da; this peptide was fragmented and 26 residues (VGGAAPGSFNTQTI/LDSPDSATFEHYY) could be determined. Using the tblastn function against filtered model transcripts Quinapyramine in the T. reesei genome (Martinez et al., 2008), the gene was found on scaffold_15: 471437–472471. An ORF encoding a protein of 344 amino acids (protein ID name 65162) with a family fh18 domain was identified (Fig. 2). The experimentally determined internal peptide sequences covered almost 33% of the Endo T sequence (underlined in Fig. 2). Some unexpected tryptic peptides
(cleavage after Q97, T280 and E290) were observed. The latter residue could represent the C-terminus of the protein, and the other unspecific cleavage sites. Analysis using the signalp web application for predicting signal sequence cleavage sites indicates a 17-amino acid signal peptide. However, because the N-terminal sequence of the Endo T protein was determined as AEPTD, nine additional residues have been cleaved off. This processed form was used for numbering in Fig. 2. Upon C-terminal sequencing, only a strong signal for a glutamate (E) residue was detected. Four potential N-glycosylation sites were present: Asn70, Asn170, Asn240 and Asn316 (Fig. 2). The electrospray ionization (ESI) mass spectrum of the purified sample showed one abundant species of 32 102 Da with no evidence for glycoforms (data not shown).