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Design of transgenes for efficient expression of active chimeric proteins on mammalian cells
Author(s) -
Liao KuangWen,
Chou WanChih,
Lo YuChih,
Roffler Steve R.
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.1064
Subject(s) - asialoglycoprotein receptor , endocytosis , chinese hamster ovary cell , chimera (genetics) , fusion protein , heterologous , microbiology and biotechnology , biology , immunotoxin , protein engineering , endoplasmic reticulum , transmembrane protein , expression vector , decay accelerating factor , chemistry , receptor , recombinant dna , hepatocyte , biochemistry , antibody , gene , in vitro , cytotoxicity , enzyme , complement system , immunology
Heterologous proteins expressed on the surface of cells may be useful for eliciting therapeutic responses and engineering new extracellular properties. We examined factors that control the membrane targeting of α‐fetoprotein (AFP) and a single‐chain antibody (scFv). Chimeric proteins were targeted to the plasma membrane by employing the transmembrane domain (TM) and cytosolic tail of murine CD8O (B7‐1), the TM of the human platelet‐derived growth factor receptor (PDGFR), the glycosylphosphatidylinositol anchor encoded by the C‐terminal extension of decay‐accelerating factor (DAF), and the TM of the H1 subunit of the human asialoglycoprotein receptor (ASGPR). AFP chimeric proteins containing the B7, DAF, ASGPR, or PDGFR targeting domains displayed half‐lives of 12.2, 3.8, 2.4, and 1.6 h, respectively. The newly synthesized B7 chimera was rapidly transported and remained on the cell surface. Glycosylphosphatidylinositol‐anchored chimeras reached the surface more slowly and significant amounts were released into the culture medium. PDGFR TM chimeras were rapidly degraded, whereas ASGPR chimeras were retained in the endoplasmic reticulum (ER). The surface expression of both AFP and scFv chimeric proteins followed the order (highest to lowest) of B7 > DAF >> PDGFR. Introduction of a dimerization domain (hinge—CH 2 —CH 3 region of human IgG1) between scFv and TM dramatically reduced cleavage of the chimeric protein, increased surface expression, and produced biologically active scFv. Our results indicate that transgenes designed for the expression of active scFv on cells should incorporate a TM that does not undergo endocytosis, include an intact cytoplasmic domain, and possess a spacer to reduce cleavage and retain biological activity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 313–323, 2001.