Supplementary Materials Supplemental Data supp_286_39_34440__index. 6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site Arranon pontent inhibitor where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an 60-fold increased rate in the protein value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed. and (18C21). The intein cleavage activities have also been developed into useful tools including the expressed protein ligation method for protein labeling/modification (22) and the Intein mediated purification with an affinity chitin-binding tag (IMPACT) Arranon pontent inhibitor method for easy purification of recombinant proteins (23). Different inteins typically exhibit different specificities for the extein amino acid residues proximal to the intein, which can severely limit the general usefulness of intein-based methods. The intein sequence plus the first C-extein residue (invariably Ser, Cys, or Thr) generally contain all the necessary structural information for protein splicing, requiring no exogenous co-factors or energy sources. However, inteins tested outside of their native host proteins often showed inefficient splicing and/or uncoupling of the splicing reaction to yield cleavage products (24C27). The selection based on the reconstitution of a selectable protein through splicing (7, 28C32) or by exploiting phage display systems (33, 34). However, in these examples, either the flanking amino acids at the splice junctions have been kept constant or only a single site was used. Thus, the identification of highly promiscuous inteins that are capable of splicing in most or ideally all sequence contexts remains an important goal. In this study, we tested whether an intein could be made more general by subjecting it to a sequential directed evolution procedure at three different insertion sites in a genetically selectable host protein. The resulting mutant inteins, when compared with the wild type intein, showed significantly improved ability of splicing at multiple KSHV ORF26 antibody new insertion sites. For one of these inteins, we also found a highly improved activity in an artificially split form that is useful for protein semisynthesis. EXPERIMENTAL PROCEDURES General Techniques Unless otherwise specified, standard protocols were used. As selection markers, kanamycin (50 g/ml) and ampicillin (100 g/ml) were applied. Synthetic oligonucleotides were obtained from Biolegio (Nijmegen, The Netherlands) and Integrated DNA Technologies (Skokie, IL). All plasmids were verified by DNA sequencing. Reagents were purchased from Acros Organics (Nidderau, Germany), Applichem (Darmstadt, Germany), GE Healthcare (Munich, Germany), Novabiochem (Bad Soden, Germany), Roth (Karlsruhe, Germany), or Sigma-Aldrich (Munich, Germany). Restriction enzymes and markers were obtained from Fermentas (St. Leon-Rot, Germany) and New England Biolabs (Ipswich, MA). All reactions and assays were performed at least in duplicate. Plasmid Construction To generate the kanamycin resistance (KanR) plasmid vector, the pDrive plasmid (Qiagen) was modified by deletion of the LacZ -peptide and multiple cloning site sequences and insertion of a His tag coding sequence at the start of the KanR gene. To facilitate intein insertion in the KanR gene, appropriate restriction sites were introduced through PCR and inverse PCR. For insertion site 1, restriction sites Arranon pontent inhibitor BspEI and SalI were inserted through silent mutations in the KanR sequence before and after the insertion site, with the corresponding amino acid sequence being -SG- and -VD-, respectively. For insertion site 2, restriction site SalI was inserted through silent mutations in the amino acid sequence -VD-, and Arranon pontent inhibitor the insertion of restriction site BglII resulted in an -SDF- to -SDL- mutation that did not prevent the KanR function. For insertion sites 3C10, restriction sites BspEI and BsrGI were Arranon pontent inhibitor inserted through silent mutations in the intein sequence, with BspEI corresponding to -SG- near the N terminus (residues 3C4) of the intein and with BsrGI corresponding to -IVH- immediately before the C terminus of the intein. Prior to directed evolution, the DnaB mini-intein coding sequence was initially amplified from pMST (35), with the appropriate restriction sites, and inserted at site 1 in the KanR plasmid vector. The plasmid pCL20 for protein.