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At least 19 recordsLinked to original sources
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↗
PubMed1996

Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process.

Protein splicing involves the self-catalyzed formation of a branched intermediate, which then resolves into the excised intervening sequence and the spliced protein. A possible mechanism for branched intermediate formation is an N-O rearrangement of the peptide bond involving the amino group of the conserved serine/cysteine residue at the upstream splice junction to yield a linear peptide ester intermediate. This possibility was examined in using an in vitro splicing system involving the intervening sequence from the DNA polymerase of the extremely thermophilic archeon, Pyrococcus sp. GB-D. Because thioesters react much more rapidly with nitrogen nucleophiles at neutral pH than do oxygen esters, protein-splicing precursors in which the serine residue of interest was replaced by cysteine were constructed and purified. In the presence of 0.25 M hydroxylamine or 0.1 M ethylene diamine at pH 6 or higher, these constructs underwent rapid cleavage at the upstream splice junction, consistent with the aminolysis of a thioester. The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage products. Comparison of the C-terminal peptide hydroxamate with the synthetic peptide hydroxamates with respect to chromatographic mobility, colorimetric assay, amino acid composition, and high-resolution mass spectrometry showed that the hydroxylamine-sensitive site in the splicing precursor was the peptide bond adjacent to the serine residue at the upstream splice junction. These results provide evidence that the peptide bond at the upstream splice junction can undergo a self-catalyzed N-O or N-S acyl rearrangement to yield a linear polypeptide ester intermediate and suggest that this kind of rearrangement constitutes the first step in protein splicing.

Amino Acid Sequence↗
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↗
PubMed1995

Protein splicing--the lengths some proteins will go to.

We review the recently discovered phenomenon of protein splicing which is the excision of an internal protein sequence at the protein level rather than at the RNA level. The means by which examples of protein splicing have been identified are described, and the similarities of the internally spliced protein products (or inteins) are discussed. Comparisons are made between inteins and group I RNA introns. We describe the evidence supporting excision of intiens by a post-translational autocatalytic reaction of a full length polypeptide precursor, rather than by RNA splicing. An examination is made of some of the proposed mechanism schemes and the supporting them presented.

Amino Acid Sequence↗
PubMed2000

Protein splicing and related forms of protein autoprocessing.

Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.

Amino Acid Sequence↗
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↗
PubMed1996

Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.

Amino Acid Sequence↗
PubMed1993

Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision.

Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino- and carboxy-terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self-excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.

Amino Acid Sequence↗
PubMed1993

Protein splicing: selfish genes invade cellular proteins.

Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.

Amino Acid Sequence↗
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↗
PubMed2004

[Protein splicing and its application in protein engineering].

Protein splicing, which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.

Exteins↗
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↗
PubMed1993

The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis.

The gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.

Amino Acid Sequence↗
PubMed1995

Protein splicing: self-splicing of genetically mobile elements at the protein level.

Protein splicing is a newly discovered process that is the protein equivalent of RNA splicing. Protein splicing proceeds through a branched protein intermediate, and in vitro studies indicate that the reaction is autocatalytic. The excised 'intein' proteins are site-specific DNA endonucleases that catalyse genetic mobility of their DNA coding sequence by an 'intein homing' mechanism.

Amino Acid Sequence↗
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↗
PubMed2000

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.

Journal Article↗
PubMed1997

Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins.

Inteins, introns spliced at the protein level, and the hedgehog family of proteins involved in eucaryotic development both undergo autocatalytic proteolysis. Here, a specific and sensitive hidden Markov model (HMM) of protein splicing domain shared by inteins and the hedgehog proteins has been trained and employed for further analysis. The HMM characterizes the common features of this domain including the position where a site-specific DNA endonuclease domain is inserted in the majority of the inteins. The HMM was used to identify several new putative inteins, such as that in the Methanococcus jannaschii klbA protein, and to generate a multiple sequence alignment of sequences possessing this domain. Phylogenetic analysis suggests that hedgehog proteins evolved from inteins. Secondary and tertiary structure predictions suggest that the domain has a structure similar to a beta-sandwich. Similarities between the serine protease cleavage mechanism and the protein splicing reaction mechanism are discussed. Examination of the locations of inteins indicates that they are not inserted randomly in an extein, but are often inserted at functionally important positions in the host proteins. A specific and sensitive HMM for a domain present in klbA proteins identified several additional bacterial and archaeal family members, and analysis of the site of insertion of the intein suggests residues that may be functionally important. This domain may play a role in formation of surface-associated protein complexes.

Algorithms↗