Design of transgenes for efficient expression of active chimeric proteins on mammalian cells

Kuang Wen Liao, Wan Chih Chou, Yu-Chih Lo, Steve R. Roffler

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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 CD80 (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 - CH2 - CH3 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.

Original languageEnglish
Pages (from-to)313-323
Number of pages11
JournalBiotechnology and Bioengineering
Volume73
Issue number4
DOIs
Publication statusPublished - 2001 May 20

Fingerprint

Platelet-Derived Growth Factor Receptors
CD55 Antigens
Asialoglycoprotein Receptor
Transgenes
Platelets
Cells
Proteins
Glycosylphosphatidylinositols
Fetal Proteins
Single-Chain Antibodies
Dimerization
Endocytosis
Endoplasmic Reticulum
Culture Media
Tail
Hinges
Cell membranes
Membrane Proteins
Bioactivity
Anchors

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Liao, Kuang Wen ; Chou, Wan Chih ; Lo, Yu-Chih ; Roffler, Steve R. / Design of transgenes for efficient expression of active chimeric proteins on mammalian cells. In: Biotechnology and Bioengineering. 2001 ; Vol. 73, No. 4. pp. 313-323.
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Design of transgenes for efficient expression of active chimeric proteins on mammalian cells. / Liao, Kuang Wen; Chou, Wan Chih; Lo, Yu-Chih; Roffler, Steve R.

In: Biotechnology and Bioengineering, Vol. 73, No. 4, 20.05.2001, p. 313-323.

Research output: Contribution to journalArticle

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