In vivo and in vitro protein solubility assays using split GFP

Abstract

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

“…The remaining 10 ␤-strands outside the ribosome give rise to an intermediate that lacks the characteristic stability of the fully folded barrel and does not support chromophore formation. These findings are consistent with split FP constructs that also fail to form a chromophore in vitro when the 11th strand is deleted (66). In contrast, the 22-aa tether (plus 11 aa C terminus) would minimally span the ribosome tunnel if fully extended.…”

Solubility-based genetic screen identifies RING finger protein 126 as an E3 ligase for activation-induced cytidine deaminase

“…Such screens include, but are not limited to, split molecule complementation (e.g., split GFP; ref. 33), chemical and photo crosslinking followed by specific immunoprecipitation to lock transient interactions in place (34), and the fusion of a bait protein with molecules that can modify and "mark" neighboring proteins (e.g., BirA; ref. 35).…”

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast ofPlasmodium falciparum

“…Additionally, a recent study in T. gondii has convincingly demonstrated a role for T. gondii Tic20 in protein transport to the apicomplexan apicoplast, a situation which is likely to also hold true for P. falciparum, thus making a role for PfsDer1-1 in this process unlikely. Future studies will aim to experimentally address the exact localization of this protein, possibly by using novel cell biological tools such as self-assembling GFP (8).…”