Preparation and characterization of human recombinant protein 1/Clara cell M(r) 10,000 protein.
Protein 1, which is identical to human Clara cell M(r) 10(4) protein, is a homodimeric, low molecular mass protein (M(r) 14,000) and an effective inhibitor of phospholipase A2 activity. We have expressed this protein in E. coli and characterized its physiochemical and biological properties. Using a pET expression system, about 1.7 mg of purified recombinant protein 1 was obtained from 250 ml of E. coli culture. The amino-terminal sequence of recombinant protein 1 up to the 20th residue was identical to that of native protein 1 except for an extra methionine at the amino-terminus. On reversed-phase HPLC, recombinant protein 1 eluted at the same retention time as native protein 1. The dose-response curves of recombinant protein 1 and native protein 1 in an enzyme-linked immunosorbent assay for protein 1 were identical. Recombinant protein 1 inhibited both porcine pancreas and cobra venom phospholipase A2 activities. These results indicated that recombinant protein 1 is structurally and biologically identical to native protein 1. We found that recombinant protein 1 also inhibits phosphatidylinositol-specific phospholipase C activity.
Okutani
R
R
Department of Clinical Pathology, Jichi Medical School, Tochigi, Japan.
Itoh
Y
Y
Yamada
T
T
Yamaguchi
T
T
Singh
G
G
Yagisawa
H
H
Kawai
T
T
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D054803:Phospholipase C delta; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014598:Uteroglobin
D007527:Isoenzymes; D011506:Proteins; D011994:Recombinant Proteins; C556389:SCGB1A1 protein, human; D014598:Uteroglobin; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D054803:Phospholipase C delta