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PubMed1997

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.

Affinity Labels↗

PubMed2003

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Versatile protein biotinylation strategies for potential high-throughput proteomics.

We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.

Biotinylation↗

PubMed2001

Reversible inhibition of protein splicing by zinc ion.

Protein splicing involves the self-catalyzed excision of a protein-splicing element, the intein, from flanking polypeptides, the exteins, which are concomitantly joined by a peptide bond. Taking advantage of recently developed in vitro systems in which protein splicing occurs in trans to assay for protein-splicing inhibitors, we discovered that low concentrations of Zn(2+) inhibited splicing mediated both by the RecA intein from Mycobacterium tuberculosis and by the naturally split DnaE intein from Synechocystis sp. PCC6803. Inhibition by Zn(2+) was also observed with a cis-splicing system involving the RecA intein. In all experimental systems used, inhibition by Zn(2+) could be completely reversed by the addition of EDTA. Zinc ion also inhibited hydroxylamine-dependent N-terminal cleavage of the RecA intein. All other divalent transition metal ions tested were less effective as inhibitors than Zn(2+). The reversible inhibition by Zn(2+) should be useful in studies of the mechanism of protein splicing and allow structural studies of unmodified protein-splicing precursors.

Bacterial Proteins↗

PubMed2003

Protein splicing of yeast VMA1-derived endonuclease via thiazolidine intermediates.

Protein splicing precisely excises out an internal intein segment from a protein precursor, and concomitantly ligates the N- and C-terminal extein polypeptides flanking the intein. A recombinant X10SNS bearing N- and C-extein polypeptides has been prepared for the intein endonuclease derived from the Saccharomyces cerevisiae VMA1 gene. X10SNS has replacements of C284S, H362N and C738S, and forms the intein and extein segments in the crystal lattice. The crystal structure of X10SNS revealed a linkage between the N- and C-extein segments, and showed that the C284 amino group of the resultant intein segment is in interaction with the G283 O atom of the N-extein segment. A mechanism for the final S --> N acyl shift step proposes that a tetrahedral intermediate involves a five-membered thiazolidine ring at G283-C738 junction. An oxyanion of the thiazolidine intermediate is to be stabilized by the C284 N atom.

Computer Simulation↗

PubMed2001

Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time.

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.

Bacterial Proteins↗

PubMed2003

An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator.

This paper describes an in vitro fluorometric assay system for protein splicing based on the RecA intein of Mycobacterium tuberculosis and a modified green fluorescent protein (GFP). The assay takes advantage of the fact that polypeptides inserted adjacent to residue 129 of GFP cause the protein to form inclusion bodies when expressed in Escherichia coli and to be incapable of fluorophore formation. However, when the inserted polypeptide is an intein, the renatured fusion protein can undergo protein splicing and chromophore formation. Comparison of chromophore formation by renatured GFP-intein fusion and renatured GFP showed that under optimal conditions (pH 6.5 and 20 degrees C) protein splicing is significantly slower than GFP chromophore formation. Taking advantage of the reversible inhibition of protein splicing by zinc ion, a fluorometric protein splicing assay was developed in which the denatured fusion protein of GFP and the RecA intein was purified on a metal ion affinity column and renatured in the presence of 2 mM ZnCl2. When diluted into appropriate buffers, protein splicing could be initiated by the addition of a molar excess of EDTA and followed fluorometrically. This assay should be valuable as a high-throughput screening system for protein splicing inhibitors as potential antimycobacterial agents and as tools for studying the mechanism of protein splicing.

Bacterial Proteins↗

PubMed2003

Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo.

Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.

Animals↗

PubMed2005

Self-cleavable stimulus responsive tags for protein purification without chromatography.

A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.

Chromatography↗

PubMed2002

Retrovirally delivered random cyclic Peptide libraries yield inhibitors of interleukin-4 signaling in human B cells.

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line epsilon transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

B-Lymphocytes↗

PubMed1998

Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.

Affinity Labels↗

PubMed1994

The ins and outs of protein splicing elements.

Protein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product, in a post-translational event analogous to the removal of an intron from rRNA. Protein splicing introns, or 'inteins' appear to be a novel type of genetic element capable of mediating gene conversion of an 'intein-less' allele, and hence promoting their own dissemination. The mechanism by which protein splicing is achieved is probably entirely encoded within the internal protein sequence, or intein, and does not require other accessory molecules. Although the concept of protein splicing inteins as selfish genetic elements of no immediate consequence to the host organism has emerged, this interpretation is questioned by recent evidence that in at least one example there appears to have been selection for protein splicing.

Amino Acid Sequence↗

PubMed2006

Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site.

VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.

Active Transport, Cell Nucleus↗

PubMed2006

Nuclear import of ho endonuclease utilizes two nuclear localization signals and four importins of the ribosomal import system.

Activity of Ho, the yeast mating switch endonuclease, is restricted to a narrow time window of the cell cycle. Ho is unstable and despite being a nuclear protein is exported to the cytoplasm for proteasomal degradation. We report here the molecular basis for the highly efficient nuclear import of Ho and the relation between its short half-life and passage through the nucleus. The Ho nuclear import machinery is functionally redundant, being based on two bipartite nuclear localization signals, recognized by four importins of the ribosomal import system. Ho degradation is regulated by the DNA damage response and Ho retained in the cytoplasm is stabilized, implying that Ho acquires its crucial degradation signals in the nucleus. Ho arose by domestication of a fungal VMA1 intein. A comparison of the primary sequences of Ho and fungal VMA1 inteins shows that the Ho nuclear localization signals are highly conserved in all Ho proteins, but are absent from VMA1 inteins. Thus adoption of a highly efficient import strategy occurred very early in the evolution of Ho. This may have been a crucial factor in establishment of homothallism in yeast, and a key event in the rise of the Saccharomyces sensu stricto.

Active Transport, Cell Nucleus↗

PubMed1999

Use of benzyl mercaptan for direct preparation of long polypeptide benzylthio esters as substrates of subtiligase.

Subtiligase, a double mutant of subtilisin, has been shown to be capable of joining together two unprotected peptide fragments, namely an activated peptide ester and a second peptide with a free N-terminal amino group. Inside cells, inteins are know to join peptide chains (exteins) by self-extrusion. The SC VMA1 intein was modified to undergo only in vitro N-terminal cleavage in the presence of small nucleophilic compounds, releasing the N-terminal extein. With a proper choice of the nucleophilic compounds it is shown that it is possible to generate long polypeptides, by molecular biology expression, with such an attached reactive ester which is an excellent substrate of the enzyme, subtiligase. This approach can successfully extend the current limit of the subtiligase-catalyzed fragment condensation method as well as provide another application of the recently discovered intein chemistry.

Benzyl Compounds↗

PubMed2002

Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides.

Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor model and is then subjected to a 2.1A resolution crystallographic analysis. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation. The S284 O(gamma) atom located at a distance of 3.1A from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S(gamma) atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of Nright arrowS acyl shift at the junction between the N-extein and intein. The S738 O(gamma) atom in the C-terminal junction is placed in close proximity to the S284 O(gamma) atom at a distance of 3.6A, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step. The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site.

Amino Acid Sequence↗

PubMed2000

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Cloning, Molecular↗

PubMed2005

Recent advances in protein splicing: manipulating proteins in vitro and in vivo.

Protein splicing is an intricate self-catalyzed protein rearrangement that converts an inactive protein precursor to biologically active proteins. In the past decade, mechanistic studies and extensive engineering of the naturally occurring protein splicing elements, termed inteins, has led to the development of numerous novel technologies. These intein-based methodologies permit in vitro and in vivo protein processing in ways previously not possible using traditional biochemical and genetic approaches. Inteins have been utilized in the production of protein and peptide arrays, as molecular switches and in the reconstitution of functional proteins by split-gene techniques.

Animals↗