Functional expression of human cathepsin S in Saccharomyces cerevisiae. Purification and characterization of the recombinant enzyme.
A cDNA encoding the human lysosomal cysteine proteinase cathepsin S precursor has been expressed in yeast using the pVT100-U expression vector containing the alpha-factor promoter. The procathepsin S gene was expressed either as a fusion protein with the pre-region or with the prepro-region of the yeast alpha-factor precursor gene. Following in vitro processing both constructs gave an identical active mature enzyme with a molecular weight of 24,000. After prolonged cultivation of the cells the recombinant protein is also found as an active proteinase in the culture supernatant. The precursor can be activated in vitro at pH 4.5 and 40 degrees C under reducing conditions. The in vitro activated enzyme has a 6-amino acid NH2-terminal extension when compared with the native bovine enzyme. The purified enzyme displays a bell-shaped pH activity profile with a pH optimum of 6.5 and pK values of 4.5 and 7.8. The isoelectric point of the recombinant human cathepsin S is between 8.3 and 8.6 and about 1.5 pH units higher than for the bovine enzyme. The kinetic data for several synthetic substrates and inhibitors reveal a preference for smaller amino acid residues in the binding subsites S2 and S3 of cathepsin S. Like the bovine enzyme, the recombinant human cathepsin S is characterized by a broader range of pH stability (pH 5-7.5) than cathepsins B and L.
Brömme
D
D
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Bonneau
P R
PR
Lachance
P
P
Wiederanders
B
B
Kirschke
H
H
Peters
C
C
Thomas
D Y
DY
Storer
A C
AC
Vernet
T
T
D001483:Base Sequence; D002403:Cathepsins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D004247:DNA; D002403:Cathepsins; C028217:cathepsin S