In vivo and in vitro protein solubility assays using split GFP

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Rapid assessment of protein solubility is crucial for evaluating proteins and variants as reagents.
• Traditional methods like antibody blots have limited capacity, and other split systems (e.g., beta-galactosidase, split GFP) can interfere with protein folding.
• A robust protein-tagging system using self-complementing split superfolder GFP (sfGFP) was previously developed to overcome these limitations.

Purpose of the Study:

• To present a detailed, step-by-step procedure for a protein solubility assay utilizing the split superfolder GFP system.
• To demonstrate the utility of this split-GFP system for high-throughput screening of protein solubility in both in vivo and in vitro formats.
• To provide a method for selecting soluble protein variants from a library.

Main Methods:

• A 15-amino-acid GFP fragment (GFP 11) is N-terminally fused to the test protein cloned into the pTET GFP 11 vector.
• The complementary GFP 1-10 detector fragment is expressed separately from the pET GFP 1-10 plasmid in Escherichia coli.
• The two fragments spontaneously associate to reconstitute a fluorescent GFP, indicating solubility of the tagged protein.

Main Results:

• The split-GFP system, comprising GFP 11 and GFP 1-10 fragments, allows for spontaneous formation of fluorescent GFP upon association.
• The GFP 11 tag exhibits minimal impact on the solubility and folding of the fused test protein.
• The assay is demonstrated to be amenable to high-throughput screening in both in vivo and in vitro formats, enabling efficient identification of soluble clones.

Conclusions:

• The described split-GFP solubility assay provides a powerful and efficient tool for identifying soluble proteins suitable for purification and downstream applications.
• The system combines the high screening capacity of in vivo assays with the accuracy of in vitro assays.
• This method offers a significant advancement for protein engineering and reagent development, overcoming limitations of previous solubility screening techniques.