Engineering inteins for protein splicing in cis and in trans by rational design and directed evolution.

摘要

Natural inteins spontaneously catalyze a protein splicing reaction to excise their own sequences and join the flanking extein sequences. By contrast, artificial inteins have been engineered to undergo controllable splicing for various practical applications. However, previously developed controllable inteins often exhibit background splicing even in the absence of inducing conditions, which thus may limit their usefulness. We have engineered two artificial inteins (the 'S10' intein and the 'S78' intein) whose activities could be controlled by intein fragment complementation. A middle fragment (of either a large or small size) of the Ssp DnaB mini-intein was deleted, and the gap was bridged with a short flexible linker sequence. The resulting incomplete intein was inactive but could be reactivated to undergo protein splicing when the missing middle fragment was supplied in trans as a separately produced protein. Under optimized conditions in vitro, the splicing efficiency of the S10 and S78 inteins reached ∼100% and ∼40%, respectively. This new method completely prevented any basal level of spontaneous splicing. This controllable intein, when inserted in target proteins, can serve as a molecular switch to control the function (maturation through splicing) of the target protein through controlling the addition ( in vitro) or expression (in vivo) of the middle fragment of the intein. In addition, since the intein sequence can be removed precisely after protein splicing, various tag or marker proteins have been inserted into the S10 and S78 inteins to create intein-tag/marker cassettes that are still capable of controllable splicing;A new method named 'internal peptide splicing' (IPS) has been invented for site-specific protein modifications by using split-inteins capable of protein trans-splicing. With two engineered split-inteins, a small peptide could be spliced into the. internal location of the target protein in a site-specific manner. This method has also been demonstrated by inserting a peptide labeled with a fluorophore into an internal site of the target protein.;Inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins in order to splice. These native extein residues remain in the spliced protein after protein splicing, and potentially affect the spliced protein function. To overcome this limitation, directed evolution was performed on the Ssp GyrB mini-intein to generate more universal inteins that can splice in different host proteins with much less reliance on specific proximal extein residues. These resulting improved inteins, when compared with the wild type intein, showed a more general ability for splicing at multiple new insertion sites of the host protein (KanR) and different host proteins.