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At least 325 records · Page 18Linked to original sources

PubMed2002

Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide.

Carrier peptides offer new opportunities to overcome problems in cellular drug delivery. Their objectives are improved cellular uptake or permeation of biological membranes, which are important pharmacokinetic features for the cellular distribution of therapeutics. Previously, human calcitonin (hCT) and selected C-terminal hCT fragments have been shown to be internalized and to permeate the epithelium of the nasal mucosa. To assess the potential of hCT-derived carrier peptides for cellular internalization of a model protein we fused enhanced green fluorescent protein (EGFP) and the [C(8)]hCT8-32 fragment by using expressed protein ligation (EPL). EGFP thioester was obtained by intein-mediated purification with an affinity chitin-binding tag (the IMPACT system, based on protein splicing). Internalization of EGFP-[C(8)]hCT8-32 by excised bovine nasal mucosa was monitored by confocal laser scanning microscopy. This novel conjugate displayed internalization into some sectors of the mucosa, whereas EGFP itself was not capable of translocation. Thus, we demonstrate successful internalization of a model protein through ligation to an hCT-derived carrier peptide, which has potential for the delivery of therapeutics. At this point the respective mechanism of translocation is unknown.

Aequorin↗

PubMed1998

Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis.

A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described. This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively. The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

The Unexpected Catalytic Properties of a Heterodimer of GAR Transformylase.

We have developed an efficient expression and purification protocol for a heterodimer of glycinamide ribonucleotide transformylase that was identified in incremental truncation libraries, a general combinatorial method for protein fragment complementation (M. Ostermeier, A. E. Nixon, J. H. Shim, and S. J. Benkovic, [1999], Proc. Natl. Acad. Sci. USA 96, 3562-3567). This heterodimer (B13) containing both a bisection point and a deletion in conserved residues close to the active site was expressed and purified in high yield using Intein methodology. The N-terminus fragment (1-111) and C-terminus fragment (M114-212) were also expressed separately as stable proteins. When these two fragments were mixed together, they associate at a highly specific 1:1 ratio to give only the active heterodimer, B13. The activity of B13 is comparable to that of the wild type and the pH-dependent kinetics of B13 turned out to be nearly identical to those of the wild type, indicating that B13 operates in the same mechanism as the wild type. This result demonstrated that cutting within conserved regions is a viable domain separation and confirmed the generality of using incremental truncation for protein fragment complementation.

Journal Article↗

PubMed1999

Expression and characterization of human salivary statherin from Escherichia coli using two different fusion constructs.

Saliva is a supersaturated solution with respect to hydroxyapatite, the main inorganic component of tooth enamel. Several acidic phosphoproteins are present in saliva which allow the supersaturated state to be maintained without random crystallization occurring. Statherin is the only salivary protein currently known to inhibit both the primary and secondary precipitation of hydroxyapatite in the supersaturated environment of saliva. To identify the residues of statherin that are necessary to control biomineralization, a recombinant form of human statherin was produced from Escherichia coli using a yeast intein fusion construct. The primary structure of the recombinant statherin was characterized by SDS-PAGE, N-terminus sequencing, MALDI mass spectrometry, and amino acid analysis and found to have the expected values relative to human-derived statherin. The secondary structure of the recombinant statherin was investigated by circular dichroism spectroscopy, which revealed the predominant presence of random coil in phosphate-buffered saline solution, with a higher propensity toward alpha helicity in 100% TFE. This increase in helicity in 100% TFE was also found in statherin that was synthesized by solid-phase synthesis. These results demonstrate that human statherin can be produced in a recombinant form which behaves comparably to the natural form.

Amino Acid Sequence↗

PubMed1999

Homing endonucleases: structure, function and evolution.

'Homing' is the lateral transfer of an intervening genetic sequence, either an intron or an intein, to a cognate allele that lacks that element. The end result of homing is the duplication of the intervening sequence. The process is initiated by site-specific endonucleases that are encoded by open reading frames within the mobile elements. Several features of these proteins make them attractive subjects for structural and functional studies. First, these endonucleases, while unique, may be contrasted with a variety of enzymes involved in nucleic acid strand breakage and rearrangement, particularly restriction endonucleases. Second, because they are encoded within the intervening sequence, there are interesting limitations on the position and length of their open reading frames, and therefore on their structures. Third, these enzymes display a unique strategy of flexible recognition of very long DNA target sites. This strategy allows these sequences to minimize nonspecific cleavage within the host genome, while maximizing the ability of the endonuclease to cleave closely related variants of the homing site. Recent studies explain a great deal about the biochemical and genetic mechanisms of homing, and also about the structure and function of several representative members of the homing endonuclease families.

Animals↗

PubMed2005

Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase.

We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.

Amino Acid Sequence↗

PubMed2001

Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747.

The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96-98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltose-binding protein (MalE) and as an intein-chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme.

ATP-Binding Cassette Transporters↗

PubMed2001

The novel function of a short region K253xRxxxD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii.

The DNA polymerase gene of the hyperthermophile Pyrococcus horikoshii was successfully overexpressed after removing an intein. The importance of an amino acid sequence around a highly conserved Asp was studied by site-directed mutagenesis. The results indicated that Lys253, Arg255, and Asp259 form a novel functional motif, K253xRxxxD259 (outside known motifs Exo I, II, and III), that is important not only for exonuclease activity but also for polymerizing activity, confirming functional interdependence between the polymerase and exonuclease domains. The short loop region, K253G254R255, probably contributes to binding to DNA substrates. Moreover, the negative charge and the side-chain length of D259 might play a supporting role in coordinating the conserved Mg2+ to the correct position at the active center in the exonuclease domain.

Amino Acid Sequence↗

PubMed2005

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli.

Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h. We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The second LLO construct contained an N-terminus His tag; it gave a yield of 3.5-8 mg l(-1). The third contained a C-terminus His tag; it gave a yield of 2.5 mg l(-1) LLO. The purified LLO from the latter two constructs retained its activity at 4 degrees C for over a year as determined by bovine red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant LLO, and is the first to provide evidence of long-term stability of the toxin for further applications.

Bacterial Toxins↗

PubMed2003

Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi.

Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi. Evidences have shown that Ca(2+) may play an important regulatory role in trypanosomatid flagellar mobility. In these parasites, the response of the cell to variations of Ca(2+) levels is determined by a variety of calcium-modulated proteins. Starting from T. cruzi cDNA lambdagt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected. rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment. In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography. Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca(2+) binding. The Ca(2+) binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca(2+)-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K(a) of 3.3+/-0.5 (10(6)) and 1.9+/-0.2 (10(4)) M(-1). Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca(2+)-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor.

Amino Acid Sequence↗

PubMed2006

Characterization of mitochondrial thioredoxin reductase from C. elegans.

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.

Animals↗

PubMed2005

Selective chemical labeling of proteins in living cells.

Labeling proteins with fluorophores, affinity labels or other chemically or optically active species is immensely useful for studying protein function in living cells or tissue. The use of genetically encoded green fluorescent protein and its variants has been particularly valuable in this regard. In an effort to increase the diversity of available protein labels, various efforts to append small molecules to selected proteins in vivo have been reported. This review discusses recent advances in selective, in vivo protein labeling based on small molecule ligand-receptor interactions, intein-mediated processes, and enzyme-catalyzed protein modifications.

Animals↗

PubMed2005

Protein semisynthesis and expressed protein ligation: chasing a protein's tail.

The adaptation of native chemical ligation to protein semisynthesis has become a powerful way to address problems in the analysis of protein structure and function. In particular, the exploitation of nature's inteins in expressed protein ligation is now a standard approach in the study of proteins. Site-specific incorporation of unnatural amino acids, biophysical probes and post-translational modifications in proteins have led to new insights into enzyme mechanisms, protein folding, ion channel function, translation and signaling.

Amino Acids↗

PubMed2006

LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression.

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP-HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT-PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT-CN system.

Amino Acid Sequence↗

PubMed2004

Isolation and characterization of new homing endonuclease specificities at individual target site positions.

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.

Base Pairing↗

PubMed2004

Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene.

To maximize spread of their host intron or intein, many homing endonucleases recognize nucleotides that code for important and conserved amino acid residues of the target gene. Here, we examine the cleavage requirements for I-TevI, which binds a stretch of thymidylate synthase (TS) DNA that codes for functionally critical residues in the TS active site. Using an in vitro selection scheme, we identified two base-pairs in the I-TevI cleavage site region as important for cleavage efficiency. These were confirmed by comparison of I-TevI cleavage efficiencies on mutant and on wild-type substrates. We also showed that nicking of the bottom strand by I-TevI is not affected by mutation of residues surrounding the bottom-strand cleavage site, unlike other homing endonucleases. One of these two base-pairs is universally conserved in all TS sequences, and is identical with a previously identified cleavage determinant of I-BmoI, a related GIY-YIG endonuclease that binds a homologous stretch of TS-encoding DNA. The other base-pair is conserved only in a subset of TS genes that includes the I-TevI, but not the I-BmoI, target sequence. Both the I-TevI and I-BmoI cleavage site requirements correspond to functionally critical residues involved in an extensive hydrogen bond network within the TS active site. Remarkably, these cleavage requirements correlate with TS phylogeny in bacteria, suggesting that each endonuclease has individually adapted to efficiently cleave distinct TS substrates.

Base Sequence↗

PubMed2005

Expression and characterization of N-terminal domain of Epstein-Barr virus latent membrane protein 2A in Escherichia coli.

Latency of Epstein-Barr virus (EBV) is maintained by the transmembrane protein latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. LMP2A contains a cytoplasmic N-terminal domain composed of 119 amino acids, which provides signals that are responsible for the association with various signal molecules, resulting in negative regulation of B-cell signaling and the EBV lytic cycle. In the present study, to obtain N-terminal domain of LMP2A (LMP2A NTD, 13 kDa) in Escherichia coli for structural analysis, a strategy for obtaining the unfused form of LMP2A NTD without any fusion partners was proposed. Recombinant LMP2A NTD has previously been expressed using the GST fusion system in E. coli [Virology 268 (2000) 178, J. Virol. 71 (1997) 4752, Mol. Cell. Biol. 20 (2000) 8526]. However, we were unable to obtain untagged LMP2A NTD from this construct because of rapid proteolysis by thrombin. To overcome the proteolysis by thrombin, C-terminal His-tagged LMP2A NTD and intein-fused LMP2A NTD were prepared. As a result, LMP2A NTD without a fusion partner could be successfully obtained using non-enzymatic cleavage. The secondary structure of the recombinant LMP2A NTD was analyzed using circular dichroism. In aqueous solution, LMP2A NTD adopts an unordered structure, which was not affected by varying pH and salt concentration. In addition, any secondary structural components of LMP2A NTD were not induced in the membrane-mimicking environments, suggesting that LMP2A NTD may intrinsically have a random coil-like structure. The biological activity of recombinant LMP2A NTD was monitored by chemical shift perturbation in HSQC spectra of LMP2A NTD with or without WW domains, which result supports that the structural change induced by WW domains is restricted within narrow region.

Amino Acid Sequence↗

PubMed2006

Cloning of Nt.CviQII nicking endonuclease and its cognate methyltransferase: M.CviQII methylates AG sequences.

Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N(6)-methyladenine). Here, we report the cloning, expression, and characterization of the NY-2A-encoded CviQII nicking-modification (N-M) system. The nicking endonuclease, Nt.CviQII, recognizes R downward arrowAG (R=A or G, downward arrow indicating cleavage site) sequences and cleaves the phosphodiester bond 5' to the adenosine. Because of the difficulty in cloning and expressing the wild-type Nt.CviQII, C-terminal truncation mutants were generated and full-length Nt.CviQII was reconstructed by intein-mediated peptide ligation. The truncation mutants and the reconstructed full-length Nt.CviQII have the same recognition and cleavage specificity as the native enzyme. Full-length and truncated Nt.CviQII produced by a cell-free transcription/translation system have similar reaction rates. The methyltransferase, M.CviQII, was also cloned and expressed. It modifies the adenine in AG doublets of DNA in vitro and in vivo in Escherichia coli. To our knowledge, M.CviQII is the first adenine methyltransferase that recognizes a dinucleotide. Therefore, M.CviQII may be a useful reagent for blocking endonuclease cleavage when restriction sites overlap with AG sequences.

Amino Acid Sequence↗