Recombinant human chymase produced by silkworm-baculovirus expression system: its application for a chymase detection kit.
Human chymase is a mast cell-derived serine proteinase, which is a non-angiotensin converting enzyme angiotensin II-generating enzyme. It appears to participate in various diseases, but it is unclear whether chymase plays major roles in physiological and pathophysiological functions in vivo. To obtain information on the physiological and pathophysiological functions of chymase and to search for diseases in which chymase participates, in the present study, we aimed at producing recombinant human chymase in large quantities and at developing an ELISA system using anti-human chymase antibodies. A recombinant human chymase was produced by a silkworm-baculovirus expression system. The recombinant chymase in active form was efficiently purified from larval hemolymph using cation-exchange and heparin column chromatography. This recombinant enzyme was enzymatically identical with native human chymase. On the other hand, the stability of the recombinant enzyme in cultured medium for mammalian cells at 37 degrees C was very high as compared with the stability of the native enzyme; 20% of the activity was maintained 120 h after addition of medium. These results indicated that the recombinant enzyme could also utilize in vitro and in vivo assay systems. We obtained several anti-chymase monoclonal antibodies by using the recombinant human chymase as antigen. These antibodies were used to construct an ELISA system for measuring the chymase concentration in blood. As a result of preliminary examination using this ELISA system, it was shown that the chymase concentration in each serum from hypertensive patients is significantly higher than in normal serum. The ELISA system will be applicable for clinical diagnosis and in vivo evaluation systems for chymase-targeting drugs.
Suzuki
Takeo
T
Chuo-Sanken Laboratory, Katakura Industries Co., Ltd., Saitama, Japan. tk.suzuki@katakura.co.jp
Kaki
Hiroki
H
Naya
Shinichi
S
Murayama
Soji
S
Tatsui
Akira
A
Nagai
Akihiko
A
Takai
Shinji
S
Miyazaki
Mizuo
M
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx / Q000378:metabolism*; D053818:Chymases; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053818:Chymases