Single-column purification of syncephapepsin--an aspartic proteinase from Syncephalastrum racemosum.
Syncephapepsin is a fungal aspartic proteinase from Syncephalastrum racemosum. By using the property of syncephapepsin after having increased activity at higher temperature, two rapid purification protocols were developed. In the former case, a crude extract was initially diluted fivefold with an activity assay buffer and heated at 50 degrees C overnight. In this situation, syncephapepsin would digest most of the proteins that the crude extract contained. Subsequently, syncephapepsin of the crude extract was precipitated from 50 to 70% of ammonium sulfate and the preparation was then directly applied to the Superdex 200 HR FPLC column. In this manner, syncephapepsin was rapidly purified to apparent homogeneity within 24 h. In this report, an alternative method of purification is also provided. Compared with the procedure mentioned above, the heating step was proceeded after FPLC chromatography through which the same result was obtained. Using cytochrome c and RNase A as substrates, the cleavage sites of both substrates were identified by HPLC peptide mapping. The results showed that syncephapepsin had a broad specificity. Residues recognized to be cleaved were primarily those of trypsin and chymotrypsin and Lys was the most susceptible.
Ho
H C
HC
School of Medicine, China Medical College, Taichung, Taiwan.
Shiau
P F
PF
Wu
S L
SL
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009090:Mucorales / Q000201:enzymology*; D012259:Ribonuclease, Pancreatic / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
D003574:Cytochrome c Group; D012259:Ribonuclease, Pancreatic; D016282:Aspartic Acid Endopeptidases; C102016:syncephapepsin; D002122:Calcium Chloride