Properties of recombinant human plasma procarboxypeptidase U produced in mammalian and insect cells.
BACKGROUND
Carboxypeptidase U (EC 3.4.17.20, TAFIa) is a new member of the metallocarboxypeptidase family circulating in human plasma as a zymogen. It is activated during coagulation and is considered as an important player in the regulation of fibrinolysis.


METHODS
Heterologous expression of human plasma procarboxypeptidase U (proCPU, TAFI) was obtained in mammalian cells (C127 and DON) and in insect cells (Sf21 and H5 cells). Conditioned media were purified by cation-exchange chromatography and plasminogen affinity chromatography to yield an essentially pure protein.


RESULTS
All systems gave high expression levels (6-20 mg/l). Due to differences in glycosylation of the activation peptide, the recombinant variants of proCPU migrated differently on SDS-PAGE (52-65 kDa). However, after activation, all active recombinant enzymes migrated at 35 kDa, similar to native CPU and no evidence for post-translational modification of the catalytic domains could be detected. For the mammalian cell produced variants, activation was more efficient after desialylation. After activation, CPU showed low solubility (0.2 mg/ml) but was inhibited similarly as native CPU.


CONCLUSIONS
Mammalian cell systems were the most efficient for the production of human plasma recombinant proCPU. The obtained zymogen differs with respect to the extent and the heterogeneity of glycosylation but, after activation, the experiments did not reveal any alteration between the recombinant and native protein.
AstraZeneca R&D, Department of Molecular Biology, SE-431 83 Mölndal, Sweden.
Mats
M
Strömqvist
Lennart
L
Hansson
Jan-Olof
JO
Andersson
Thord
T
Johansson
Michael
M
Edlund
Maria
M
Enoksson
Filip
F
Goossens
Simon
S
Scharpé
Dirk
D
Hendriks
D000818:Animals; D025901:Carboxypeptidase B2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000494:pharmacology*; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000097:blood; D006031:Glycosylation; D006801:Humans; D007313:Insecta / Q000378:metabolism*; D007525:Isoelectric Focusing; D037102:Lectins; D008322:Mammals / Q000378:metabolism; D013058:Mass Spectrometry; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D004792:Enzyme Precursors; D037102:Lectins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D025901:Carboxypeptidase B2