Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.
We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.
Yonemura
Hiroshi
H
Applied Research Department, The Chemo-Sero-Therapeutic Research Institute (KAKETSUKEN), Kawabe, Kyokushi, Kikuchi, Kumamoto 869-1298.
Imamura
Takayuki
T
Soejima
Kenji
K
Nakahara
Yo
Y
Morikawa
Wataru
W
Ushio
Yoshitaka
Y
Kamachi
Yasuharu
Y
Nakatake
Hiroshi
H
Sugawara
Keishin
K
Nakagaki
Tomohiro
T
Nozaki
Chikateru
C
D000199:Actins / Q000378:metabolism; D000818:Animals; D001709:Biotechnology / Q000379:methods; D015153:Blotting, Western; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D002846:Chromatography, Affinity; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009101:Multiple Myeloma / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D010974:Platelet Aggregation; D011401:Promoter Regions, Genetic; D011516:Prothrombin / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000199:Actins; D018076:DNA, Complementary; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D011516:Prothrombin; D012685:Sepharose; C012007:prethrombins; D013762:Tetrahydrofolate Dehydrogenase; D010450:Endopeptidases; D013917:Thrombin; C020097:ecarin; D008727:Methotrexate