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At least 271 records · Page 15Linked to original sources

PubMed2005

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation.

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.

Base Sequence↗

PubMed2004

An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

Animals↗

PubMed2005

Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum.

The type III secretion system (YscC) protein of Yersinia pestis plays an essential role in the translocation of Yersinia outer proteins (Yops) into eukaryotic target cells through a type III secretion mechanism. To assess the immunogenicity and potential protective efficacy of YscC against lethal plague challenge, we cloned, overexpressed, and purified YscC using two different bacterial expression and purification systems. The resulting expression plasmids for YscC, pETBlue-2-YscC and pTYB11-YscC, were regulated by robust T7 promoters that were induced with isopropyl-beta-D-thiogalactopyranoside. The intein-fusion pTYB11-YscC system and the six-histidine-tagging pETBlue-2-YscC system were both successful for producing and purifying YscC. The intein-mediated purification system produced about 1mg of soluble YscC per liter of bacterial culture while the YscC-His(6)-tag method resulted in 16mg of insoluble YscC per liter of bacterial culture. Protein identity for purified YscC-His(6) was confirmed by ion trap mass spectrometry. Antisera were produced against both YscC and YscC-His(6). The specific immune response generated in YscC-vaccinated mice was relative to the particular purified protein, YscC or YscC-His(6), which was used for vaccination as determined by Western blot analysis and ELISA. Regardless of the purification method, either form of the YscC protein failed to elicit a protective immune response against lethal plague challenge with either F1 capsule forming Y. pestis CO92 or the isogenic F1(-)Y. pestis C12.

Amino Acid Sequence↗

PubMed2005

Expression of compstatin in Escherichia coli: incorporation of unnatural amino acids enhances its activity.

Compstatin, a 13-residue cyclic peptide, is a complement inhibitor that shows therapeutic potential. Several previous approaches have improved the activity of this peptide several-fold. In the present study, we have expressed and purified compstatin from Escherichia coli in an effort to increase its potency and to generate it in high yield in a more economical fashion. An intein-based expression system was used to express compstatin in fusion with chitin-binding domain and Ssp DnaB intein, which were later cleaved from the expressed molecule at room temperature and pH 7.0 to yield pure compstatin in one step. The expressed compstatin showed activity similar to the synthetic compstatin in an ELISA-based assay. The same expression system and purification strategy were used to incorporate three tryptophan analogs, 6-fluoro-tryptophan, 5-hydroxy-tryptophan, and 7-aza-tryptophan, into compstatin. Interestingly, incorporation of 6-fluoro-tryptophan increased the activity three-fold relative to wild-type compstatin; in contrast, incorporation of 5-hydroxy- or 7-aza-tryptophan rendered compstatin less active than the wild-type form.

Amino Acid Sequence↗

PubMed2006

One-step purification of insoluble hydantoinase overproduced in Escherichia coli.

Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B11291 (HDTar) results in the formation of insoluble aggregates in Escherichia coli. As previously reported, recombinant HDTar could be obtained in a homogeneous form using one chromatographic step. However, soluble proteins are required for the pre-treatment in several steps before proceeding to the chromatographic purification step. In this study, we reported a method based on artificial oil bodies (AOBs) to obtain homologous HDTar from its insoluble form in one step. By linkage of HDTar to intein-oleosin gene fusion, the tripartite fusion protein was over-expressed as aggregates in E. coli. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. Further induction for peptide cleavage mediated by intein, the bound HDTar was liberated from AOBs, and the protein free of fusion tags was then recovered. As a result, refolded HDTar was amplified by over 300-fold. Obviously, this simplified method provides an efficient way to obtain HDTar with high yield and high purity.

Amidohydrolases↗

PubMed1997

Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins.

The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.

Amino Acid Sequence↗

PubMed2000

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.

Biotechnology↗

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↗

PubMed2002

Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag.

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 degrees C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly-Arg-Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies.

Affinity Labels↗

PubMed2001

Structural requirements for the biosynthesis of backbone cyclic peptide libraries.

BACKGROUND: Combinatorial methods for the production of molecular libraries are an important source of ligand diversity for chemical biology. Synthetic methods focus on the production of small molecules that must traverse the cell membrane to elicit a response. Genetic methods enable intracellular ligand production, but products must typically be large molecules in order to withstand cellular catabolism. Here we describe an intein-based approach to biosynthesis of backbone cyclic peptide libraries that combines the strengths of synthetic and genetic methods. RESULTS: Through site-directed mutagenesis we show that the DnaE intein from Synechocystis sp. PCC6803 is very promiscuous with respect to peptide substrate composition, and can generate cyclic products ranging from four to nine amino acids. Libraries with five variable amino acids and either one or four fixed residues were prepared, yielding between 10(7) and 10(8) transformants. The majority of randomly selected clones from each library gave cyclic products. CONCLUSIONS: We have developed a versatile method for producing intracellular libraries of small, stable cyclic peptides. Genetic encoding enables facile manipulation of vast numbers of compounds, while low molecular weight ensures ready pharmacophore identification. The demonstrated flexibility of the method towards both peptide length and composition makes it a valuable addition to existing methods for generating ligand diversity.

Amino Acid Sequence↗

PubMed2005

Homing endonuclease structure and function.

Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.

Alternative Splicing↗

PubMed2002

Semisynthesis and folding of the potassium channel KcsA.

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.

Amino Acid Sequence↗

PubMed2003

Protein semi-synthesis in living cells.

Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. Here we describe an approach that allows ligation of synthetic molecules to target proteins in an intracellular environment. A cellular protein is genetically tagged with one-half of a split intein. The complementary half is linked in vitro to the synthetic probe, and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein trans-splicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes.

Animals↗

PubMed1998

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination.

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.

Alleles↗

PubMed1994

A proposed mechanism for the self-splicing of proteins.

Intervening protein sequences, called inteins, are intronlike elements that are removed posttranslationally, apparently by self-splicing. The conserved and essential residues of precursor proteins consist of an asparagine as the last residue of the intein and a hydroxyl- or thiol-containing residue immediately following both splice junctions. Evidence for a branched intermediate has been reported [Xu, M.-Q., Southworth, M., Mersha, F., Hornstra, L. & Perler, F. (1993) Cell 75, 1371-1377]; however, the chemical nature of the branched structure is unclear. I propose a mechanism that includes the formation of a branched structure, provides an explanation for the reversal of branch formation observed at high pH, and accounts for each of the essential amino acids. The branched structure is formed by nucleophilic attack of the asparagine side chain on the N-terminal splice junction. The nature of this branched structure is a distinguishing feature of the model and can be experimentally tested.

Asparagine↗

PubMed1996

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Amino Acid Sequence↗

PubMed1999

Production of cyclic peptides and proteins in vivo.

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

Bacterial Proteins↗

PubMed1999

Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.

Animals↗