Selection for intrabody solubility in mammalian cells using GFP fusions

Area of Science:

• Biotechnology
• Molecular Biology
• Proteomics

Background:

• Single-chain antibody fragments (scFv), known as intrabodies, are valuable tools in functional proteomics.
• However, their utility is often hindered by aggregation issues within the cytoplasm of mammalian cells.

Purpose of the Study:

• To investigate the differential behavior of homologous scFv in mammalian cells.
• To establish a method for evaluating and selecting soluble intrabodies for mammalian expression.

Main Methods:

• Comparison of two homologous scFv with differing aggregation properties in E. coli and mammalian cells.
• Expression of scFv as fusions with Green Fluorescent Protein (GFP) using retroviral vectors at low levels.
• Utilizing GFP fluorescence as a proxy for soluble intrabody expression in mammalian cells.
• Application of the GFP-based assay to screen an E. coli-optimized intrabody library.

Main Results:

• One scFv aggregated in mammalian cells, while a homologous scFv remained soluble and active.
• Low-level expression with GFP fusion revealed that aggregated scFv were degraded, leading to low fluorescence, whereas soluble scFv produced high fluorescence.
• The GFP+ cell population successfully enriched for soluble mammalian intrabodies.
• Demonstrated distinct requirements for soluble intrabody expression between E. coli and mammalian systems.

Conclusions:

• Green Fluorescent Protein (GFP) signal intensity can effectively predict intrabody solubility in mammalian cells.
• Intrabody libraries can be directly optimized for mammalian expression using a straightforward GFP-based screening assay.
• This method facilitates the development of functional intrabodies for applications in human cells.