Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins

Abstract

Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport. In PGAP2 mutant cells, most GPI-APs are secreted into the medium. Here, we established a HEK293 cell line where endogenous PGAP2 was knocked out and exogenous PGAP2 was inserted with a piggyBac transposon in the genome. Using these cells, human lysosomal acid lipase (LIPA) and α-galactosidase A (GLA) were expressed as GPI-anchored forms (LIPA-GPI and GLA-GPI) and cells expressing high levels of LIPA-GPI or GLA-GPI on the cell surface were enriched. Removal of the PGAP2 gene by piggyBac transposase or FLP recombinase converted LIPA-GPI and GLA-GPI from membrane-bound to the secreted forms. Thus, cells expressing LIPA or GLA in large amounts could be enriched using this approach. The GPI-based molecular switching system is an efficient approach to isolate cells expressing recombinant proteins with high productivity.

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

“…As elegantly demonstrated by Matabaro et al . 5 , a GPI-anchored protein used for cell selection can be switched to a secreted form that in case of gp41 peptide may enhance its effectiveness by providing protection to non-edited cells. Other SORTS applications that can be envisioned include monitoring of promoter activity or inactivation of non-coding RNAs 41 .…”

“…Here, we report the development of a new strategy called Surface Oligopeptide knock-in for Rapid Target Selection (SORTS) that enables the sorting of edited cells via knock-in (KI) of a short genetic element encoding an epitope targeted to the cell surface via a GPI anchor 5,6 and designed to inactivate the start codon of the targeted gene (Fig. 1a).…”

Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41

“…We reasoned that the antiviral activity of gp41 peptides can be expanded from gene-modified cells to bystander cells by replacing the GPI-anchored form of a peptide with a soluble one. Matabaro et al ( 43 ) demonstrated that by regulating the fatty acid remodeling of GPI-anchored proteins, it is possible to achieve partial membrane expression of a protein needed for fluorescence-activated cell sorting (FACS) as well as protein secretion. Another interesting approach for enhancing the activity of an anti-HIV inhibitory peptide is noncovalently linking it to a CXCR4 or CCR5 coreceptor, as demonstrated for the 15D peptide ( 44 ).…”

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells

“…So, then the target protein could be expressed in the cell membrane to sort the highly expressed cells. Finally, the target protein can be secreted into the culture for subsequent isolation and purification after removing the PGAP2 gene by adding the transposase again ( Matabaro et al, 2017 ). The flexible use of the transposon system’s ability to both integrate and shear genes allows researchers to artificially regulate whether target proteins are secreted or not.…”