DNA encoding the human serine protease C-E

Abstract

Here we describe the molecular identification of a cDNA encoding a novel serine protease we have termed protease C-E. The deduced amino acid sequence, and it alignment with other well-characterized serine proteases indicates that it is a member of the S1 serine protease family. We have found that the protease C-E mRNA is expressed in pancreas, placenta, prostate, small intestine, stomach, spleen, fibroblasts and epidermis, as well as in certain regions of the brain i.e., cerebellum, cerebral cortex, pituitary and hippocampus. Enzymatically active protease C-E, as produced using the methodologies described herein, is amenable to further biochemical analyses for the identification of physiological substrates and specific modulators.

Description

BACKGROUND OF THE INVENTION

[0001] The members of the trypsin / chymotrypsin-like (S1) serine protease family are gaining recognition due to the increased awareness that these enzymes play pivotal roles in a multitude of diverse physiological processes. In addition to the classical functions the proteases trypsin and chymotrypsin perform during the digestive process, serine proteases also participate in regulating key amplification cascades through the proteolytic activation of inactive zymogen precursors. Thus, in many instances the protease substrates within these cascades are themselves the inactive form, or zymogen, of a “downstream” serine protease. Well-known examples of this serine protease-mediated regulation include blood coagulation, (Davie et al. (1991). Biochemistry 30:10363-70), kinin formation (Proud and Kaplan (1988). Annu. Rev. Immunol. 6:49-83) and the complement system (Reid and Porter (1981). Annual Review of Biochemistry 50:433-464). Although these proteolytic path...

Examples

example 1

Plasmid Manipulations:

[0128] All molecular biological methods were in accordance with those previously described (Maniatis et al. (1989). 1-1626). Oligonucleotides were purchased from Ransom Hill Biosciences (Ransom Hill, Calif.) and all restriction endonucleases and other DNA modifying enzymes were from New England Biolabs (Beverly, Mass.) unless otherwise specified. The protease C-E expression construct was made in the baculovirus expression vector pFastBac1 (Life Technologies, Gaithersberg, Md.) as described below. All construct manipulations were confirmed by dye terminator cycle sequencing using Allied Biosystems 377 fluorescent sequencers (Perkin Elmer, Foster City, Calif.).

Acquisition of Protease C-E cDNA

[0129] Primers were designed to flank the putative open reading frame and used in a

SEQ.ID.NO.:3.:C-E F / L-U 5′-GGATAAAACCTGGGGGGACCTG-3′SEQ.ID.NO.:4:C-E F / L-L 5′-TCCGGGGCCCCAGAGGTAGATGAG-3′

preparative PCR reaction using human prostate marathon ready cDNA (Clontech, Pal...

BACKGROUND OF THE INVENTION

[0001] The members of the trypsin / chymotrypsin-like (S1) serine protease family are gaining recognition due to the increased awareness that these enzymes play pivotal roles in a multitude of diverse physiological processes. In addition to the classical functions the proteases trypsin and chymotrypsin perform during the digestive process, serine proteases also participate in regulating key amplification cascades through the proteolytic activation of inactive zymogen precursors. Thus, in many instances the protease substrates within these cascades are themselves the inactive form, or zymogen, of a “downstream” serine protease. Well-known examples of this serine protease-mediated regulation include blood coagulation, (Davie et al. (1991). Biochemistry 30:10363-70), kinin formation (Proud and Kaplan (1988). Annu. Rev. Immunol. 6:49-83) and the complement system (Reid and Porter (1981). Annual Review of Biochemistry 50:433-464). Although these proteolytic path...

Examples

example 1

Plasmid Manipulations:

[0128] All molecular biological methods were in accordance with those previously described (Maniatis et al. (1989). 1-1626). Oligonucleotides were purchased from Ransom Hill Biosciences (Ransom Hill, Calif.) and all restriction endonucleases and other DNA modifying enzymes were from New England Biolabs (Beverly, Mass.) unless otherwise specified. The protease C-E expression construct was made in the baculovirus expression vector pFastBac1 (Life Technologies, Gaithersberg, Md.) as described below. All construct manipulations were confirmed by dye terminator cycle sequencing using Allied Biosystems 377 fluorescent sequencers (Perkin Elmer, Foster City, Calif.).

Acquisition of Protease C-E cDNA

[0129] Primers were designed to flank the putative open reading frame and used in a

SEQ.ID.NO.:3.:C-E F / L-U 5′-GGATAAAACCTGGGGGGACCTG-3′SEQ.ID.NO.:4:C-E F / L-L 5′-TCCGGGGCCCCAGAGGTAGATGAG-3′

preparative PCR reaction using human prostate marathon ready cDNA (Clontech, Pal...

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