Conditional Protein Splicing:  A New Tool to Control Protein Structure and Function in Vitro and in Vivo

Abstract

Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.

Enigmatic Distribution, Evolution, and Function of Inteins

“…For example, the activity of a trans-splicing intein (Fig. 1B) has been manipulated to depend on the presence of the small molecule, rapamycin, that induces intein reassociation and splicing (86,87). Another way to control the intein relies on the substitution of the HEN domain by a receptor and utilization of a receptor-ligand binding event to trigger splicing.…”

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

“…Protein splicing in trans could ligate two foreign peptide chains that are fused with either N-or C-terminal fragments (N-or C-inteins) of a split intein [4][5][6]. Protein trans-splicing system has opened many applications including segmental isotopic labelling of proteins, protein cyclization, in vivo protein engineering, and site-specific chemical modifications [6][7][8][9][10][11][12][13][14][15]. However, protein ligation by protein trans-splicing can be significantly modulated by the junction sequences as well as by the extein sequences, which could limit the applications of protein trans-splicing [16].…”

A small-molecule switch for Golgi sulfotransferases

“…2E, both reconstituted enzymes showed dose-dependent activity characterized by EC 50 values of 0.8 nM for GlcNAc6ST-1 and 0.1 nM for GlcNAc6ST-2. The specificity of the response was confirmed by competition experiments with the drug ascomycin, which binds to FKBP but not to FRB and thus prevents formation of the rapamycin͞FKBP͞FRB complex (15,36). Ascomycin inhibited the rapamycin-dependent response of both sulfotransferases in a dose-dependent manner (Fig.…”