Function of the propeptide region in recombinant expression of active procathepsin L in Escherichia coli.
In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.
Ogino
T
T
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, 569-1094, Japan.
Kaji
T
T
Kawabata
M
M
Satoh
K
K
Tomoo
K
K
Ishida
T
T
Yamazaki
H
H
Ishidoh
K
K
Kominami
E
E
D000818:Animals; D020134:Catalytic Domain; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L