Francine B. Perler

SINGLE-COLUMN PURIFICATION OF FREE RECOMBINANT PROTEINS USING A SELF-CLEAVABLE AFFINITY TAG DERIVED FROM A PROTEIN SPLICING ELEMENT

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

InBase, the Intein Database

Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliography. An intein-specific BLAST server is now available to assist in identifying new inteins.

Protein Splicing of Inteins and Hedgehog Autoproteolysis: Structure, Function, and Evolution

The Mxe GyrA mini-intein represents a splicing element without a DRR or core endonuclease domain. Larger inteins, like the Sce VMA intein, contain homing endonucleases. The structural similarity among splicing elements and Hh-C17 can be seen in the common positioning of β strands and the large number of residues with superimposable Cα atoms (the main chain carbon atom to which the side chain is attached) (Figure 3Figure 3). However, there is little sequence identity among the three proteins except for the residues involved in catalysis (Figure 1Figure 1 and Figure 3Figure 3). Because of the similarity of their core structures, Hall et al. 1997xHall, T.M.T, Porter, J.A, Young, K.E, Koonin, E.V, Beachy, P.A, and Leahy, D.J. Cell. 1997; 91: 85–97Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all ReferencesHall et al. 1997 proposed that inteins and Hh-C17 evolved from a common precursor (Figure 4Figure 4). The Hint module mediates ester/thioester formation, activating the linkage between the element and a second protein domain. Other systems, such as the Ntn hydrolase family, employ an acyl rearrangement to activate catalysis but do not have structural similarity to the Hint module (Hall et al. 1997xHall, T.M.T, Porter, J.A, Young, K.E, Koonin, E.V, Beachy, P.A, and Leahy, D.J. Cell. 1997; 91: 85–97Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all ReferencesHall et al. 1997). Inteins and Hh-C subsequently evolved separate methods of cleaving this bond. Inteins evolved the ability to ligate two exteins and acquired the DRR and core endonuclease. The endonuclease allowed inteins to spread by lateral transmission. Hh-C17 acquired a sterol recognition region (SRR) directing addition of cholesterol to the hedgehog protein signaling domain, anchoring the signaling domain to the cell surface. Alternatively, the Hint module could have invaded a preexisting signaling domain/SRR element. Several nematode Hh-C domains contain unrelated C-terminal extensions that may interact with molecules other than cholesterol and have been tentatively termed adduct recognition regions (Beachy et al. 1997xCold Spring Harbor Symp. Beachy, P.A, Cooper, M.K, Young, K.E, von Kessler, D.P, Park, W, Hall, T.M.T, Leahy, D.J, and Porter, J.A. Quant. Biol. 1997; in pressSee all ReferencesBeachy et al. 1997). The order of these events is speculative, including the order of module assembly. Each of the intein and hedgehog elements may have coevolved or associated after independent formation. Endonucleases may have also been lost from inteins. As we understand the mechanism of protein splicing and Hh-C autoproteolysis, we will begin to be able to harness these elements to cleave or splice any target protein at will.Figure 4A Scenario for Evolution of Inteins and Hh-CA primordial subdomain was duplicated and loop exchange occurred between subdomains to generate the Hint module. Inteins then acquired the DNA recognition region (DRR) and core endonuclease domain (ENDO) and Hh-C17 acquired a sterol recognition region (SRR). The order of these events is speculative. Adapted with permission from Hall et al., 1997.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Dissecting the Chemistry of Protein Splicing and Its Applications

Protein splicing, the protein equivalent of RNA splicing, is a newly discovered posttranslational process that proceeds through a branched protein intermediate and produces two separate polypeptides from one gene. The experimental data used to distinguish among the proposed protein-splicing mechanisms are presented along with the progress made towards fully describing the mechanism. Numerous protein engineering applications using modified inteins have been developed, including the generation of alpha-thioesters in proteins, which circumvent the limits of solid-phase peptide synthesis.

In vitro protein splicing of purified precursor and the identification of a branched intermediate

Protein splicing is a posttranslational processing event in which an internal polypeptide is excised from a protein precursor and the terminal polypeptides are then ligated together, resulting in the production of two proteins. This report presents direct evidence for protein splicing by demonstrating in vitro splicing of purified precursor that accumulated when the protein splicing element from Pyrococcus DNA polymerase was cloned into a foreign gene. In vitro splicing was temperature and pH dependent. A slowly migrating species exhibited kinetic properties of a splicing intermediate and was shown to be a branched molecule by N-terminal sequencing. The precursor and slowly migrating species were interconvertible in response to pH shifts.

Control of protein splicing by intein fragment reassembly.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol‐1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans‐splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.

Protein splicing and autoproteolysis mechanisms

It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.

Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C‐terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali‐labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.

An alternative protein splicing mechanism for inteins lacking an N‐terminal nucleophile

Variations in the intein‐mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N‐terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N‐terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N‐terminal splice junction (thio)ester. Despite the presence of an N‐terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non‐canonical pathway, the C‐extein nucleophile attacks a peptide bond at the N‐terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N‐terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.

Protein Splicing and its Applications

Protein splicing elements, termed inteins, provide a fertile source for innovative biotechnology tools. First harnessed for protein purification, inteins are now used to express cytotoxic proteins, to segmentally modify or label proteins, to cyclize proteins or peptides, to study structure-activity relationships and to generate reactive polypeptide termini in expressed proteins for an expanding list of chemoselective reactions, including protein ligation.