Direct expression of recombinant activated human protein C, a serine protease.
Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens.
Ehrlich
H J
HJ
Lilly Research Laboratories, Indianapolis, Indiana 46285.
Jaskunas
S R
SR
Grinnell
B W
BW
Yan
S B
SB
Bang
N U
NU
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011486:Protein C / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D000925:Anticoagulants; D011486:Protein C; D011994:Recombinant Proteins; D012697:Serine Endopeptidases