Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector.
The subtilisin-related proprotein convertase furin is expressed in various mammalian tissues. Expecting that COS-1 cells have a furin-like endoprotease, we constructed a fusion expression vector for production of a recombinant foreign protein having no signal peptide or a protein in truncated form into secreted mature protein. A cDNA fragment encoding N-terminal procalcitonin (pro-CT) of human calcitonin precursor was inserted into the mammalian expression vector pME18S. We used PCR techniques to generate four kinds of cDNAs encoding the C terminus of the pro-CT with Arg residues at P4 (Arg-Xaa-Lys-Arg), P6 (Arg-Xaa-Xaa-Xaa-Lys-Arg), or both (Arg-Xaa-Arg-Xaa-Lys-Arg), in addition to the Lys-Arg motif at the cleavage site, in order to determine the conditions for efficient processing in nonendocrine cells, such as COS-1 cells. The cDNA coding for the Fc fragment of human immunoglobulin G1 was fused in-frame to the cDNA encoding pro-CT at its C terminus. Upon transfection of the chimeric plasmids into COS-1 cells, almost all of the fusion protein with the Arg residues at both P4 and P6 were processed into secreted Fc product, even without cotransfection of furin. These results indicate that COS-1 cells have a furin-like endoprotease and suggest that pro-CT, with the Arg residues at both P4 and P6, can be used as a carrier peptide for expression of a foreign protein having no signal peptide or a protein in truncated form in COS-1 cells.
Liu
Y C
YC
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Maebashi, Japan.
Kawagishi
M
M
Mikayama
T
T
Inagaki
Y
Y
Takeuchi
T
T
Ohashi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002116:Calcitonin / Q000235:genetics; D015740:Calcitonin Gene-Related Peptide; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D045683:Furin; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D007668:Kidney; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011498:Protein Precursors / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D015183:Restriction Mapping; D013381:Subtilisins / Q000096:biosynthesis* / Q000378:metabolism; D014162:Transfection; D001743:Urinary Bladder
C000606589:CALCA protein, human; D017931:DNA Primers; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002116:Calcitonin; D013381:Subtilisins; D045683:Furin; D015740:Calcitonin Gene-Related Peptide