Characterization of cathepsin L secreted by Sf21 insect cells.
Sf21 cells, derived from the Spodoptera frugiperda pupa, are commonly used for the heterologous expression of proteins. While purifying recombinant proteins from this system we encountered a protease, secreted at high levels by Sf21 cells, that readily degraded recombinant proteins and also tended to co-purify with histidine-tagged proteins from Ni(2+) affinity columns. Purification and characterization of the protease revealed that it has many properties consistent with cysteine proteases of the papain family, including autoactivation under reducing conditions and acidic pH, and inhibition by E-64. Amino acid sequence analysis showed that the Sf21 enzyme may be identical to a putative insect procathepsin L cloned from the cotton bollworm. The subsite specificity of the Sf21 cathepsin and its inhibition profile by cystatins are consistent with the protease being an insect homologue of cathepsin L. Monoclonal antibodies useful for the detection and purification of the insect cathepsin L were developed.
Johnson
Gary D
GD
R&D Systems, Inc. 614 McKinley Pl. NE, Minneapolis, MN 55413, USA. garyj@rndsystems.com
Jiang
Weiping
W
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D015853:Cysteine Proteinase Inhibitors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; D007930:Leucine; C024974:E 64