A general purification platform for toxic proteins based on intein trans-splicing

Abstract

Many important functional proteins often exhibit toxicity when overexpressed in heterologous hosts. Unfortunately, this toxicity can complicate the production of these proteins in recombinant systems, which can slow their characterization. Although a number of engineered expression strains and plasmids have been developed to optimize toxic protein expression, many targets remain recalcitrant in these systems due to extreme toxicity to the expression host. In this work, we have developed a novel protein purification platform based on intein trans-splicing, with special relevance for proteins that are extremely toxic to recombinant host cells. The toxic protein is split into two inactive fragments, which are separately expressed in fusion to the segments of a split intein. The N-terminal intein segment is first immobilized onto an affinity column and washed, followed by addition of the C-terminal segment and purification of the complex. The assembled intein controllably splices to deliver the mature target protein, simultaneously releasing the purified target from the affinity column. To optimize this method, we generated a hybrid split intein consisting of the N-terminus of the Npu DnaE intein and the C-terminus of the Ssp DnaE intein. This hybrid intein tolerates a wider range of amino acids at the +2 site of the C-terminal splicing junction than the Npu intein alone. In the production of the highly toxic homing endonuclease I-TevI, the yield from the hybrid intein is 50 % higher than the native Npu DnaE intein, while the I-TevI protein purified from both inteins showed native activity.

Improved protein splicing using embedded split inteins

“…Naturally split inteins are widely employed in protein engineering studies. Applications include the segmental isotopic labeling of proteins, the generation of toxic proteins and the semisynthesis of proteins . Despite the broad utility of PTS, the approach can suffer from the poor behavior of the reactive polypeptides.…”

“…Applications include the segmental isotopic labeling of proteins, the generation of toxic proteins and the semisynthesis of proteins. 18,19 Despite the broad utility of PTS, the approach can suffer from the poor behavior of the reactive polypeptides. This is a particular problem for constructs containing the Int N fragment due to its partially disordered structure.…”

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

“…Some demonstrated strategies have included modulation of split intein association, reversible inhibition by metal ions, ligand induced structural rearrangements, salt induction, and direct chemical modification of catalytic residues (Buskirk et al, 2004;Ciragan, Aranko, Tascon, & Iwai, 2016;Lew & Paulus, 2002;Shi, Tarimala, Meng, & Wood, 2014;Skretas & Wood, 2005;Wong, Mosabbir, & Truong, 2015;Wood & Camarero, 2014). Some demonstrated strategies have included modulation of split intein association, reversible inhibition by metal ions, ligand induced structural rearrangements, salt induction, and direct chemical modification of catalytic residues (Buskirk et al, 2004;Ciragan, Aranko, Tascon, & Iwai, 2016;Lew & Paulus, 2002;Shi, Tarimala, Meng, & Wood, 2014;Skretas & Wood, 2005;Wong, Mosabbir, & Truong, 2015;Wood & Camarero, 2014).…”

“…The primary challenge facing intein application is spacio-temporal control of the typically autocatalytic splicing or cleavage activity. Some demonstrated strategies have included modulation of split intein association, reversible inhibition by metal ions, ligand induced structural rearrangements, salt induction, and direct chemical modification of catalytic residues (Buskirk et al, 2004;Ciragan, Aranko, Tascon, & Iwai, 2016;Lew & Paulus, 2002;Shi, Tarimala, Meng, & Wood, 2014;Skretas & Wood, 2005;Wong, Mosabbir, & Truong, 2015;Wood & Camarero, 2014). For applications involving N-terminal cleavage this chemical modification is typically performed on the attacking nucleophile at the first position of the intein.…”

Inteins as tools for tagless and traceless protein purification

“…One alternative is to use the splicing activity of split inteins to simultaneously assemble and purify the target proteins, where splicing, rather than cleaving, releases the target from the tag. Although this approach is largely unexplored for most applications, it has been demonstrated for the production of highly toxic proteins . Another alternative, however, is the development of engineered inteins with unusual sensitivities, which might provide the controllable and rapid cleaving required.…”