The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleavi...

Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-fr...

Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems. To overcome this difficulty, we have established a novel approach, termed the intein-mediated modulation of protein stability (imPS), in which a destabilizing domain and part of a split intein are fused to the essential protein. The growth of the mutant virus c...

THY Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same allelic family as TLI Pol-2 (PI-TLII), Tfu Pol-2 (PI-TFUII) and TspTY Pol-3 mini-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intein was cloned, expressed in Escherichia coli and purified. The intein is a specific endonuclease (PI-THY:I) which cleaves the inteinless sequence of the THY D...

Protein splicing is a remarkable post-translational modification in which an intervening sequence (intein) excises itself from the host precursor protein, concomitantly ligating the two flanking sequences (exteins) with a peptide bond (Fig. 1a)1–3. Currently, more than 500 inteins have been identified in all three kingdoms of life (eukaryotes, eubacteria and archaea) on the basis of conserved c...

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water...

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scis...

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein ...

Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA polymerase II from Methanoculleus marisnigri (Mma) can promote protein splicing. However, protein splicing can be prevented or reduced by overexpression under nonreducing conditions because of the format...

Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is activ...