Active recombinant rat dipeptidyl aminopeptidase I (cathepsin C) produced using the baculovirus expression system.
An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC). In vivo activation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5 degrees C for 18-40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide-p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.
Lauritzen
C
C
UNIZYME Laboratories, Dr. Neergaards Vej 17, Horsholm, DK-2970, Denmark. mail@unizyme.dk
Pedersen
J
J
Madsen
M T
MT
Justesen
J
J
Martensen
P M
PM
Dahl
S W
SW
D000818:Animals; D020789:Cathepsin C; D002460:Cell Line; D002846:Chromatography, Affinity; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000096:biosynthesis* / Q000235:genetics; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010455:Peptides / Q000737:chemistry; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; C033223:polyhistidine; D006639:Histidine; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C