Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease.
We have constructed and optimized a high yielding Escherichia coli expression system to produce glycosylation-free human procathepsin K and have developed conditions for refolding this enzyme. Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E. coli, refolded from inclusion bodies, and further purified by Superdex 75 size-exclusion chromatography. Purified procathepsin K had a [MH]+ of 35,063 Da which is in agreement with the predicted mass of the construct. Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected. Purified procathepsin K activated under autocatalytic conditions to a final specific activity of 23 micromol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin. This expression and refolding procedure yielded 50 mg of purified, glycosylation-free human procathepsin K from 1 liter of E. coli cell culture and enabled the determination of the structure of human procathepsin K at 2.6 A resolution.
D'alessio
K J
KJ
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. Karla_J_D'Alessio@sbphrd.com
McQueney
M S
MS
Brun
K A
KA
Orsini
M J
MJ
Debouck
C M
CM
D000595:Amino Acid Sequence; D056657:Cathepsin K; D002403:Cathepsins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003374:Coumarins / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000201:enzymology*; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D003374:Coumarins; D004151:Dipeptides; D005456:Fluorescent Dyes; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K