Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins Adam M. Kerrigan, Taryn L. Powers, Deirdre M. Dorval, Julie N. Reitter, Kenneth V. Mills * Department of Chemistry, College of the Holy Cross, Worcester, MA 01610, USA article info Article history: Received 22 June 2009 Available online 3 July 2009 Keywords: Protein splicing Intein Extein Pyrococcus abyssi Ribonucleotide reductase Homing endonuclease abstract An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme ther- mophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facil- itate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins. Ó 2009 Elsevier Inc. All rights reserved. Introduction Protein splicing is the process by which an intervening polypep- tide, called an intein, facilitates its excision from flanking se- quences, the exteins, concomitant with the ligation of the exteins. This converts the precursor fusion protein (NIC) to linked exteins (NC) and excised intein (I). The standard mechanism occurs in four steps [1]. The first is an amide to ester or thioester rearrangement of the peptide bond link- ing the N-terminal extein (N-extein) to the intein, which has an N- terminal Cys or Ser. The second step is a transesterification, where- by the N-extein is transferred to the side chain of the N-terminal residue of the C-terminal extein (C-extein), a Cys, Ser or Thr. This results in a branched ester intermediate. The third step is cleavage of the intein from the linked exteins via cyclization of the con- served C-terminal Asn of the intein. Finally, the ester linking the exteins is converted to a peptide bond, and the C-terminal amino- succinimide of the intein may be hydrolyzed to Asn or iso-Asn. If the order of the steps is disrupted, usually due to mutation of conserved residues, side reactions may occur [1]. If the third step is slowed or prevented, the linear ester from step one or the branched ester from step two may undergo hydrolysis or thiolysis. This pro- cess, N-terminal cleavage, results in release of the N-extein (N) from the linked intein and C-extein (IC) fragment. If the first or sec- ond step is slowed or prevented, the third step may become uncou- pled from splicing. This process, called C-terminal cleavage, releases the C-extein (C) from the linked N-extein and intein (NI). For numbering, the first residue of the intein is designated residue 1, the first residue of the C-extein is C+1, and the final res- idue of the N-extein is N-1, with numbering proceeding upstream [2]. The alpha subunit of the ribonucleotide reductase (RIR) of the extreme thermophile Pyrococcus abyssi is interrupted by three sep- arate inteins (Fig. 1) [3,4]. We show that each intein is capable of facilitating efficient protein splicing when over-expressed in Esch- erichia coli as a fusion protein between affinity domains serving as exteins. We study the flexibility of the insertion sites of the inteins by substituting residues at the splice junctions of each intein and studying the influence of these mutations on splicing efficiency. Materials and methods Plasmid preparation. To amplify the intein genes via PCR, P. abys- si was cultured and genomic DNA isolated as described [5]. Intein RIR1 was amplified with primers RIR1U (5 0 -TGAAAAGGCCTCACG ATGTTG) and RIR1L (5 0 -GGCTGCCCCATCGATTGTTC), intein RIR2 with RIR2U (5 0 -CATGCATGCAACAACTACGGG) and RIR2L (5 0 -CACTA ACGGCATCGATGAGG), and intein RIR3 with RIR3U (5 0 -GCAAAGGG AAGGCCTATAAG) and RIR3L (5 0 -ATGGCTTCCCGTTATCGATGT). To express a fusion protein of E. coli maltose binding protein (MBP) with each intein and a poly-His tag, plasmids pMIHRIR1, pMIHRIR2, and pMIHRIR3 were created by digesting the PCR prod- ucts above with StuI and ClaI (for RIR1 and RIR3) or SphI and ClaI (for RIR2), and ligating the product of the digestion into the same sites in plasmid pPabPol1His, described previously [5]. To increase the rate of cell growth during expression and the amount of expression, mutations were made to conserved homing 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.145 * Corresponding author. Fax: +1 508 793 3530. E-mail address: kmills@holycross.eduw (K.V. Mills). Biochemical and Biophysical Research Communications 387 (2009) 153–157 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc