Purification and characterization of recombinant human cathepsin E expressed in human kidney cell line 293.
A cDNA encoding human prepro-cathepsin E was introduced into the adenovirus-transformed HEK-293 (human embryonic kidney) cell line. The construct contained both a V5 peptide epitope and histidine tags at the carboxy terminus. Transfected cells efficiently secreted recombinant pro-cathepsin E into the culture medium. The secreted pro-cathepsin E was purified in a single step using Ni affinity chromatography yielding a protein of about 92 kDa under non-reducing conditions. The amino-terminal sequence of the purified protein began at Ser20, suggesting human cathepsin E accumulated in the culture supernatant as the pro-enzyme. The purified protein was rapidly and completely converted to the active form by treatment at pH 4.0 or below. Steady state kinetic parameters for hydrolysis of the fluorogenic peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 (cleavage at the Phe-Phe bond) were consistent with previously reported values for purified human enzyme (kc/Ki= 53 x 10(6) M(-1) s(-1), Km= 6.3 microM, and kcat= 3 x 10(2) s(-1)). The activated protein was potently inhibited by pepstatin with Ki= 0.2 nM, as well as a reported beta secretase inhibitor. This work demonstrates the potential for producing large quantities of highly purified human cathepsin E from HEK-293 cells in quantities to support both biochemical and structural characterization of the enzyme.
Cappiello
Maria G
MG
Discovery Biology, Concurrent Pharmaceuticals, 502 W Office Center Drive, Fort Washington, PA 19034, USA.
Wu
Zhongren
Z
Scott
Boyd B
BB
McGeehan
Gerard M
GM
Harrison
Richard K
RK
D000595:Amino Acid Sequence; D020059:Cathepsin E / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D002403:Cathepsins; C067581:procathepsin E; D020059:Cathepsin E