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PubMed2002

Production of a recombinant antimicrobial peptide in transgenic plants using a modified VMA intein expression system.

Tobacco plants were engineered to express SMAP-29, a mammalian antimicrobial peptide of innate immunity, as fusion protein with modified vacuolar membrane ATPase intein. The peptide was purified taking advantage of the intein-mediated self-cleaving mechanism. SMAP-29 was immunologically detected in the chromatographic eluate and appeared tightly bound to copurified plant proteins. Electrophoretic separation under disaggregating conditions indicated that the recombinant peptide was cleaved off by intein at the expected site and an overlay gel assay demonstrated that the peptide retained antimicrobial activity. These results indicate that a modified intein expression system can be used to produce pharmaceutical peptides in transgenic plants.

Blood Proteins↗

PubMed2003

Distribution and function of new bacterial intein-like protein domains.

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Amino Acid Sequence↗

PubMed1997

Identification of an unusual intein in chloroplast ClpP protease of Chlamydomonas eugametos.

The proteasome-like ClpP protease is widely distributed and structurally conserved among bacteria and eukaryotic cell organelles. In Chlamydomonas eugametos, however, the chloroplast clpP gene predicted a much larger ClpP protein containing large insertion sequences (ISs). One insertion sequence, IS2, is 456 amino acid residues long and not similar to known proteins. Here we show that IS2 is an unusual intein, and its protein splicing activity in Escherichia coli cells can be activated by a single amino acid substitution. Analysis of IS2 sequence revealed short sequence motifs that are similar to known intein motifs, including putative LAGLI-DADG endonuclease motifs. But a histidine residue conserved at the C terminus of known inteins is replaced in the IS2 sequence by a glycine residue (Gly455), rendering the IS2 sequence incapable of detectable protein splicing when tested in E. coli cells. Changing Gly455 to histidine activated the ability of IS2 to undergo protein splicing in E. coli cells. The IS2 sequence (intein) was precisely excised from a precursor protein, with the flanking sequences (exteins) joined together by a normal peptide bond.

Adenosine Triphosphatases↗

PubMed1999

InBase, the New England Biolabs Intein Database.

Inteins are intervening sequences that splice as proteins, not RNA. InBase, the New England Biolabs Intein Database (http://www.neb. com/neb/inteins.html), is a comprehensive on-line database that includes the Intein Registry, along with detailed information about each intein and its host protein, tabulated comparisons and a comprehensive bibliography including papers in press.

Animals↗

PubMed2000

InBase, the Intein Database.

InBase, the Intein Database (http://www.neb.com/neb/inteins.html ), is a comprehensive on-line resource that includes the Intein Registry. Inteins are protein splicing elements that mediate a self-catalytic protein splicing reaction. InBase presents general information as well as detailed data for each intein, including tabu-lated comparisons and a comprehensive bibliography.

Databases, Factual↗

PubMed2004

Molecular mechanism of vde-initiated intein homing in yeast nuclear genome.

In Saccharomyces cerevisiae, VMAI intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMAI intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spollp-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDEinitiated homing in yeast cells.

Journal Article↗

PubMed2004

Molecular mechanism of VDE-initiated intein homing in yeast nuclear genome.

In Saccharomyces cerevisiae, VMA1 intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMA1 intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spo11p-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDE-initiated homing in yeast cells.

Biological Transport↗

PubMed2006

Reactivity of intein thioesters: appending a functional group to a protein.

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide-alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site-the C terminus-of virtually any protein.

Amines↗

PubMed2006

Expression of N-terminal Cys-protein fragments using an intein refolding strategy.

The inclusion body expression and refolding of a pH-sensitive intein fusion protein (Ssp DnaB intein) delivered sufficient quantities of an N-terminal Cys-polypeptide for native chemical ligations. This strategy circumvents premature intein cleavage under expression conditions and allows the expression and purification of proteins with uncertain solubility properties. The expressed protein resembles the C-terminal portion of the amphiphilic immunity protein Im7, which can be ligated to synthetic thioesters to yield synthetic protein analogues for protein folding studies.

Bacterial Proteins↗

PubMed2006

Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme.

Protein trans-splicing by the naturally split intein of the gene dnaE from Nostoc punctiforme (Npu DnaE) was demonstrated here with non-native exteins in Escherichia coli. Npu DnaE possesses robust trans-splicing activity with an efficiency of > 98%, which is superior to that of the DnaE intein from Synechocystis sp. strain PCC6803 (Ssp DnaE). Both the N- and C-terminal parts of the split Npu DnaE intein can be substituted with the corresponding fragment of Ssp DnaE without loss of trans-splicing activity. Protein splicing with the Npu DnaEN is also more tolerant of amino acid substitutions in the C-terminal extein sequence.

Amino Acid Sequence↗

PubMed2005

Production of native protein by using Synechocystis sp. PCC6803 DnaB mini-intein in Escherichia coli.

To directly express native recombinant proteins in Escherichia coli, a new expression vector pSB was constructed using Ssp DnaB mini-intein. Using the vector, native proteins could be produced with the help of C-terminal self-cleavage of the intein. In this study, we cloned hIFNalpha-4 gene into pSB and used E. coli strain Origami B (DE3) as the host. Expression experiments were carried out both in Shake flasks and a 5 L bioreactor. The results indicated hIFNalpha-4 could be expressed in the form of soluble protein with correct folding in E. coli. The maximal hIFNalpha-4 content was 21.7% of total protein, and the antiviral activity of the protein was 1.2x10(8 )IU mg(-1). Overall, good effects were achieved with this system. This intein-mediated protein expression system opens up a useful method for production of native recombinant protein in E. coli.

Antiviral Agents↗

PubMed2004

New type of polyubiquitin-like genes with intein-like autoprocessing domains.

Genome analysis of ciliates identified a new type of polyubiquitin-like genes. These contain tandem repeats of ubiquitin-like domains interspersed with autocatalytic intein-like domains. Inteins and related protein domains post-translationally process their own precursor proteins by protein-splicing, cleavage and ligation reactions. The structure of these polyubiquitin-like genes suggests their precursor products undergo maturation and conjugation in cis. This novel gene structure also illustrates the genetic modularity of ubiquitin-like and intein-like domains. Our suggested autoprocessing of ubiquitin-like polyproteins is a new potential general way for controlling protein functions.

Amino Acid Sequence↗

PubMed2005

Development of a tandem protein trans-splicing system based on native and engineered split inteins.

Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chemical biology and biotechnology, precious little is known about the molecular recognition events governing the initial fragment association step. In this study, fluorescence approaches have been used to measure the dissociation constant for the Ssp DnaE split intein interaction and to determine the on and off rates of fragment association. The DnaE fragments bind with low nanomolar affinity, and our data suggest that electrostatics make an important contribution to the very rapid association of the fragments at physiological pH. This information was used to develop a tandem trans-splicing system based on native and engineered split inteins. This novel system allows the one-pot assembly of three polypeptides under native conditions and can be performed in crude cell lysates. The technology should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins.

Amino Acid Sequence↗

PubMed1999

A genetic system yields self-cleaving inteins for bioseparations.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Amino Acid Sequence↗

PubMed2001

A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli.

The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale. Many peptides need carboxy amidation for full activity or prolonged bioavailability. However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity. Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system. This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents. Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide. With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.

Amides↗

PubMed1998

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se.

Bacterial Proteins↗

PubMed2004

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction.

Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The (1)J(NC') coupling constant for the (-1) scissile peptide bond at the N-extein-intein junction was found to be approximately 12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (-1) scissile bond in its unusual conformation. Overall, these studies support the "ground-state destabilization" model as part of the mechanism of catalysis.

Isotopes↗

PubMed1998

Introns and intein coding sequence in the ribonucleotide reductase genes of Bacillus subtilis temperate bacteriophage SPbeta.

The two putative ribonucleotide reductase subunits of the Bacillus subtilis bacteriophage SPbeta are encoded by the bnrdE and bnrdF genes that are highly similar to corresponding host paralogs, located on the opposite replication arm. In contrast to their bacterial counterparts, bnrdE and bnrdF each are interrupted by a group I intron, efficiently removed in vivo by mRNA processing. The bnrdF intron contains an ORF encoding a polypeptide similar to homing endonucleases responsible for intron mobility, whereas the bnrdE intron has no obvious trace of coding sequence. The downstream bnrdE exon harbors an intervening sequence not excised at the level of the primary transcript, which encodes an in-frame polypeptide displaying all the features of an intein. Presently, this is the only intein identified in bacteriophages. In addition, bnrdE provides an example of a group I intron and an intein coding sequence within the same gene.

Amino Acid Sequence↗