title: 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.
authors.affiliation: Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, 201620, People's Republic of China.
authors.forename: Changhua
authors.initials: C
authors.lastname: Shi
authors.forename: Anirudh
authors.initials: A
authors.lastname: Tarimala
authors.forename: Qing
authors.initials: Q
authors.lastname: Meng
authors.forename: David W
authors.initials: DW
authors.lastname: Wood
mesh_terms: D047668:Inteins / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity*; D020040:Trans-Splicing
chemical_list: D011994:Recombinant Proteins