PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Inteins”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources
PubMed2005

Inteins and affinity resin substitutes for protein purification and scale up.

The development of self-cleaving fusion-tag technology has greatly simplified the purification of recombinant proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods at a variety of scales. In this review, we describe some of these methods, and provide a rudimentary economic analysis of hypothetical large-scale applications. This work is expected to provide a rough outline for the evaluation of these methods for large-scale bioprocessing of a variety of products.

Journal Article↗
PubMed2005

Protein splicing mechanisms and applications.

Inteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches.

Animals↗
PubMed1996

Homing events in the gyrA gene of some mycobacteria.

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

Amino Acid Sequence↗
PubMed2001

Degeneration of a homing endonuclease and its target sequence in a wild yeast strain.

Mobile introns and inteins self-propagate by 'homing', a gene conversion process initiated by site-specific homing endonucleases. The VMA intein, which encodes the PI-SceI endonuclease in Saccharomyces cerevisiae, is present in several different yeast strains. Surprisingly, a wild wine yeast (DH1-1A) contains not only the intein(+) allele, but also an inteinless allele that has not undergone gene conversion. To elucidate how these two alleles co-exist, we characterized the endonuclease encoded by the DH1-1A intein(+) allele and the target site in the intein(-) allele. Sequence analysis reveals seven mutations in the 31 bp recognition sequence, none of which occurs at positions that are individually critical for activity. However, binding and cleavage of the sequence by PI-SceI is reduced 10-fold compared to the S.cerevisiae target. The PI-SceI analog encoded by the DH1-1A intein(+) allele contains 11 mutations at residues in the endonuclease and protein splicing domains. None affects protein splicing, but one, a R417Q substitution, accounts for most of the decrease in DNA cleavage and DNA binding activity of the DH1-1A protein. Loss of activity in the DH1-1A endonuclease and target site provides one explanation for co-existence of the intein(+) and intein(-) alleles.

Alleles↗
PubMed2004

[Construction and expression of vector with aroA-in gene and its transformation in tobacco].

Inteins are intervening protein sequences that undergo self-excision from precursor protein with concomitant joining of flanking sequences. Here, we demonstrated intein cis-splicing in Nicotiana tabacum nuclear genomes by using artificial cis Ssp DnaB and Rma DnaB intein. We want to test whether protein splicing can occur in higher eucaryotic cell,which would play an important role in transgene containment in transgenic plants. Glyphosate-resistant Salmonella typhimurium aroA gene was divided at position 235/236 aa within EPSPS by inverse PCR from pLEPSPS. Amplified gene products with artificial cis-Ssp DnaB/Rma DnaB intein and split-Ssp DnaB/Rma DnaB intein were inserted at position 235 of EPSPS respectively to construct plasmid pLEBC, pLERC, pLEBT and pLERT. Above four aroA-In gene fusions were ligated into pET-32 to obtain E. coli expression vectors termed pETLEBC, pETLEBT, pETLERC and pETLERT. E. coli DE3 cells containing individual recombinant plasmids described above were induced by IPTG to produce corresponding protein products. Detectable spliced EPSPS and unspliced precursor demonstrated that splicing occurred in bacteria. aroA-cis SSp DnaB and aroA-cis Rma DnaB were ligated into Agrobacterium tumefaciens binary vector pLYM. Then A. tumefaciens containing EPSPS-(cis) intein cassettes were used for leaf disk transformation in N. tabacum. Integration of aroA-In gene into plant genome was confirmed by genomic PCR analyses. To verify the expression of fusion genes at transcriptional level, RT-PCR analyses were performed and the expected products were identified. These results suggested that plant cells support expression of S. typhimurium aroA-In fusion gene in nulear genomes. Thus,we speculated the existence of protein-splicing activity in plant cells. This opens the possibility of applying intein trans-splicing technique to reduce/prevent gene transfer by way of pollen in transgenic plants.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2006

Protein splicing in cis and in trans.

Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallographic study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were determined using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.

Inteins↗
PubMed2005

Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

Bacillus subtilis↗
PubMed2003

[Protein splicing and its application].

Protein splicing is a newly discovered posttranslational editing process that removes an internal protein fragment from the protein precursor. During the splicing process the internal protein fragment, intein, triggered the self-excision from the precursor protein and the concomitant ligation of the flanking protein fragments, exteins. The self-catalysis requires neither auxiliary enzymes nor cofactors and only involves four intramolecular reactions. A number of key catalytic residues in inteins and flanking fragments have been identified, which led to the development of the protein splicing process as a protein engineering tool. Controllable cleavage of the peptide bond at either the N or the C terminus of an intein has allowed the design of novel strategies for manipulation of protein and peptides. Affinity purification of recombinant proteins can be facilitated by fusion the target protein with an intein. The fusion also creates C-terminal thioester, which expands the scope of chemical ligation in protein. Inteins can be engineered in a "split and inverted" configuration to form a cyclic polypeptide consisting of the sequence linking two intein subdomains. This article summarizes the recent advance in the mechanism of protein splicing and its applications in protein purification, protein ligation and protein cyclization.

Inteins↗
PubMedDate not supplied

[Protein splicing].

Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.

Inteins↗
PubMed2000

A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing.

We describe a new method with general applicability for monitoring any protein-protein interaction in vivo. The principle is based on a protein splicing system, which involves a self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to formation of a new protein in which the exteins are linked directly by a peptide bond. As the exteins, split N- and C-terminal halves of enhanced green fluorescent protein (EGFP) were used. When a single peptide consisting of an intein derived from Saccharomyces cerevisiae intervening the split EGFP was expressed in Escherichia coli, the two external regions of EGFP were ligated, thereby forming the EGFP corresponding fluorophore. Genetic alteration of the intein, which involved large deletion of the central region encoding 104 amino acids, was performed. In the expression of the residual N- and C-terminal intein fragments each fused to the split EGFP exteins, the splicing in trans did not proceed. However, upon coexpression of calmodulin and its target peptide M13, each connected to the N- and C-terminal inteins, fluorescence of EGFP was observed. These results demonstrate that interaction of calmodulin and M13 triggers the refolding of intein, which induces the protein splicing, thereby folding the ligated extein correctly for yielding the EGFP fluorophore. This method opens a new way not only to screen protein-protein interactions but also to visualize the interaction in vivo in transgenic animals.

Calmodulin↗
PubMed2004

Directed evolution of ligand dependence: small-molecule-activated protein splicing.

Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.

Amebicides↗
PubMed2003

Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes.

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with concomitant joining of the flanking sequences. Here, we demonstrate intein trans-splicing in Nicotiana tabacum chloroplasts by using the naturally split Ssp DnaE intein. Trans-splicing occurred whether both intein fragments were encoded in the chloroplast or were separated into the chloroplast and nuclear genomes. A biolistic approach was used to integrate two fusion genes, one encoding aminoglycoside-3-adenyltransferase (aadA) and the first 123 aa of the Ssp DnaE intein (In) and the other encoding 36 C-terminal amino acid residues of the Ssp DnaE intein (Ic) and soluble modified green fluorescent protein (smGFP) into N. tabacum plastids. Expression of these gene fragments in the chloroplast resulted in ligated aadA-smGFP due to In-Ic-mediated trans-splicing. Furthermore, an N-terminal portion of the herbicide resistance gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) containing a chloroplast localization signal fused to In (EPSPSn-In) was integrated into the nuclear DNA of N. tabacum by using Agrobacterium tumefaciens-mediated transformation. The remaining EPSPS gene fragment (EPSPSc) fused to Ic (Ic-EPSPSc) was integrated into the chloroplast genome by homologous recombination. Western blot analysis of cell extracts from these plants showed a full-length EPSPS, demonstrating that the EPSPSn-In gene product migrated to the chloroplast and underwent trans-splicing. Furthermore, these transgenic plants displayed improved resistance to the herbicide N-(phosphonomethyl)glycine (glyphosate) when compared with wild-type N. tabacum.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2003

Structural and functional characteristics of homing endonucleases.

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the "homing" process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.

Amino Acid Motifs↗
PubMed2005

Protein splicing elements and plants: from transgene containment to protein purification.

Protein splicing elements, termed inteins, have been discovered in all the domains of life. Basic research on inteins has led to a greater understanding of how they mediate the protein splicing process. Because inteins are natural protein engineering elements they have been harnessed for use in a number of applications, including protein purification, protein semisynthesis, and in vivo and in vitro protein modifications. This review focuses on the use of inteins in plants. A split-gene technique utilizes inteins to reconstitute the activity of a transgene product with the goal of limiting the spread of transgenes from a genetically modified plant to a weedy relative. Furthermore, merging the intein tag for protein purification with the large protein yields possible with plants has the potential to produce pharmaceutically important proteins. Finally, relevant techniques that may be used in plants in the future are discussed.

Acetolactate Synthase↗
PubMed2001

Overexpression and purification of Helicobacter pylori flavodoxin and induction of a specific antiserum in rabbits.

Flavodoxin from the gastric pathogen Helicobacter pylori has been shown to be the electron acceptor of the essential pyruvate-oxidoreductase enzyme complex and proposed to be involved in the pathogenesis of gastric MALToma. In order to obtain a sufficient amount for biochemical and structural studies, we overexpressed the protein either with a C-terminal His(6) -tag or as a fusion protein upstream of intein- and chitin-binding domains. With both expression systems we succeeded at purifying soluble and functional flavodoxin containing the cofactor FMN. When expressing with a His(6) -tag, we purified approximately 20 mg flavodoxin per liter of bacterial culture, while expression as an intein-CBD fusion protein with autocatalytic removal of the intein-CBD part rendered only approximately 1 mg of purified flavodoxin per liter of bacterial culture. Expressed as an intein-CBD fusion protein, flavodoxin copurified with a C-terminal degradation product, which was not observed for expression with a His(6) -tag. However, we were able to obtain protein crystals suited for X-ray structure determination from flavodoxin expressed as an intein-CBD fusion protein, but not from flavodoxin expressed with a C-terminal His(6) -tag. We further report the induction of a rabbit antiserum specific for H. pylori flavodoxin.

Animals↗
PubMed2000

Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139).

Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step. This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene. The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT). The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene. The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column. The recombinant plasmid was transfected into E. coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy. hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response. hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86). Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. These results indicate that the added peptides modulate cell growth by acting at the cell surface. Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.

Breast Neoplasms↗