PROCESSING OF A FUSION PROTEIN BY ENDOPROTEASE IN COS-1 CELLS FOR SECRETION OF MATURE PEPTIDE BY USING A CHIMERIC EXPRESSION VECTOR

被引:47
作者
LIU, YC
KAWAGISHI, M
MIKAYAMA, T
INAGAKI, Y
TAKEUCHI, T
OHASHI, H
机构
[1] LA JOLLA INST ALLERGY & IMMUNOL,LA JOLLA,CA 92037
[2] GUNMA UNIV,INST ENDOCRINOL,MAEBASHI,GUNMA 371,JAPAN
关键词
FURIN; CALCITONIN PRECURSOR; FC FRAGMENT;
D O I
10.1073/pnas.90.19.8957
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:8957 / 8961
页数:5
相关论文
共 27 条
  • [1] MAMMALIAN SUBTILISINS - THE LONG-SOUGHT DIBASIC PROCESSING ENDOPROTEASES
    BARR, PJ
    [J]. CELL, 1991, 66 (01) : 1 - 3
  • [2] PC1 AND PC2 ARE PROPROTEIN CONVERTASES CAPABLE OF CLEAVING PROOPIOMELANOCORTIN AT DISTINCT PAIRS OF BASIC RESIDUES
    BENJANNET, S
    RONDEAU, N
    DAY, R
    CHRETIEN, M
    SEIDAH, NG
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (09) : 3564 - 3568
  • [3] HUMAN FUR GENE ENCODES A YEAST KEX2-LIKE ENDOPROTEASE THAT CLEAVES PRO-BETA-NGF INVIVO
    BRESNAHAN, PA
    LEDUC, R
    THOMAS, L
    THORNER, J
    GIBSON, HL
    BRAKE, AJ
    BARR, PJ
    THOMAS, G
    [J]. JOURNAL OF CELL BIOLOGY, 1990, 111 (06) : 2851 - 2859
  • [4] A NEURO-ENDOCRINE PEPTIDE DERIVED FROM THE AMINO-TERMINAL HALF OF RAT PROCALCITONIN
    BURNS, DM
    BIRNBAUM, RS
    ROOS, BA
    [J]. MOLECULAR ENDOCRINOLOGY, 1989, 3 (01) : 140 - 147
  • [5] THE NUCLEOTIDE-SEQUENCE OF A HUMAN IMMUNOGLOBULIN-CGAMMA-1 GENE
    ELLISON, JW
    BERSON, BJ
    HOOD, LE
    [J]. NUCLEIC ACIDS RESEARCH, 1982, 10 (13) : 4071 - 4079
  • [6] INTRACELLULAR TARGETING AND STRUCTURAL CONSERVATION OF A PROHORMONE-PROCESSING ENDOPROTEASE
    FULLER, RS
    BRAKE, AJ
    THORNER, J
    [J]. SCIENCE, 1989, 246 (4929) : 482 - 486
  • [7] HATUSZAWA K, 1990, J BIOL CHEM, V265, P22075
  • [8] HOSAKA M, 1991, J BIOL CHEM, V266, P12127
  • [9] MICROINJECTED XENOPUS OOCYTES SECRETE MATURE, BIOLOGICALLY-ACTIVE PARATHYROID-HORMONE
    IKEDA, K
    POSILLICO, JT
    BOYLE, MB
    WEBB, AC
    BROWN, EM
    BROADUS, AE
    [J]. MOLECULAR ENDOCRINOLOGY, 1989, 3 (07) : 1084 - 1089
  • [10] IDENTIFICATION OF A 2ND HUMAN SUBTILISIN-LIKE PROTEASE GENE IN THE FES/FPS REGION OF CHROMOSOME 15
    KIEFER, MC
    TUCKER, JE
    JOH, R
    LANDSBERG, KE
    SALTMAN, D
    BARR, PJ
    [J]. DNA AND CELL BIOLOGY, 1991, 10 (10) : 757 - 769