Showing papers in "Nature Biotechnology in 1999"

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Abstract: We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we com- pared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively glob- al protein expression in cells and tissues.

A generic protein purification method for protein complex characterization and proteome exploration.

Abstract: We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein The TAP method has been tested in yeast but should be applicable to other cells or organisms

Direct analysis of protein complexes using mass spectrometry

Abstract: We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor

Abstract: Plant productivity is greatly affected by environmental stresses such as drought, salt loading, and freezing. We reported previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Fluorescent proteins from nonbioluminescent Anthozoa species.

Abstract: We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.

Abstract: We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.

A microfabricated fluorescence-activated cell sorter

Abstract: We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (µFACS) for sorting various biological entities Compared with conventional FACS machines, the µFACS provides higher sensitivity, no cross-contamination, and lower cost We have used µFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E coli cells and shown that the bacteria are viable after extraction from the sorting device These sorters can function as stand-alone devices or as components of an integrated microanalytical chip

Production of goats by somatic cell nuclear transfer

Abstract: In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity

Abstract: The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline-regulated expression of beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region-associated, bisected complex oligosaccharides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.

Rapid protein-folding assay using green fluorescent protein.

Abstract: Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.

Detection of PCR products using self-probing amplicons and fluorescence.

Abstract: Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.

Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.

Abstract: Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.

Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices

Abstract: Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices

Purification of recombinant proteins by fusion with thermally-responsive polypeptides

Abstract: Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.

De novo reconstitution of a functional mammalian urinary bladder by tissue engineering.

Abstract: Human organ replacement is limited by a donor shortage, problems with tissue compatibility, and rejection. Creation of an organ with autologous tissue would be advantageous. In this study, transplantable urinary bladder neo-organs were reproducibly created in vitro from urothelial and smooth muscle cells grown in culture from canine native bladder biopsies and seeded onto preformed bladder-shaped polymers. The native bladders were subsequently excised from canine donors and replaced with the tissue-engineered neo-organs. In functional evaluations for up to 11 months, the bladder neo-organs demonstrated a normal capacity to retain urine, normal elastic properties, and histologic architecture. This study demonstrates, for the first time, that successful reconstitution of an autonomous hollow organ is possible using tissue-engineering methods.

Iron fortification of rice seed by the soybean ferritin gene.

Abstract: To improve the iron content of rice, we have transferred the entire coding sequence of the soybean ferritin gene into Oryza sativa (L. cv. Kita-ake) by Agrobacterium-mediated transformation. The rice seed-storage protein glutelin promoter, GluB-1, was used to drive expression of the soybean gene specifically in developing, self-pollinated seeds (T1 seeds) of transgenic plants, as confirmed by reverse transcription PCR analysis. Stable accumulation of the ferritin subunit in the rice seed was demonstrated by western blot analysis, and its specific accumulation in the endosperm by immunologic tissue printing. The iron content of T1 seeds was as much as threefold greater than that of their untransformed counterparts.

Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees

Abstract: Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.

DNA delivery from polymer matrices for tissue engineering.

Abstract: We have proposed engineering tissues by the incorporation and sustained release of plasmids encoding tissue-inductive proteins from polymer matrices. Matrices of poly(lactide-co-glycolide) (PLG) were loaded with plasmid, which was subsequently released over a period ranging from days to a month in vitro. Sustained delivery of plasmid DNA from matrices led to the transfection of large numbers of cells. Furthermore, in vivo delivery of a plasmid encoding platelet-derived growth factor enhanced matrix deposition and blood vessel formation in the developing tissue. This contrasts with direct injection of the plasmid, which did not significantly affect tissue formation. This method of DNA delivery may find utility in tissue engineering and gene therapy applications.

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Abstract: Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Tumor targeting with a selective gelatinase inhibitor

Abstract: Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies

Advances in vaccine adjuvants.

Abstract: Currently, aluminum salts and MF59 are the only vaccine adjuvants approved for human use. With the development of new-generation vaccines (including recombinant subunit and mucosal vaccines) that are less immunogenic, the search for more potent vaccine adjuvants has intensified. Of the novel compounds recently evaluated in human trials, immunostimulatory molecules such as the lipopolysaccharide derived MPL and the saponin derivative QS21 appear most promising, although doubts have been raised as to their safety in humans. Preclinical work with particulate adjuvants, such as the MF59 microemulsion and lipid–particle immune-stimulating complexes (Iscoms), suggest that these molecules are also potent elicitors of humoral and cellular immune responses. In addition, preclinical data on CpG oligonucleotides appear to be encouraging, particularly with respect to their ability to selectively manipulate immune responses. While all these adjuvants show promise, further work is needed to better define the mechanisms of adjuvant action. Ultimately, the development of more potent adjuvants may allow vaccines to be used as therapeutic, rather than prophylactic, agents.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

Abstract: A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue

Abstract: We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo

Abstract: Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

Therapeutic antibody fragments with prolonged in vivo half-lives.

Abstract: Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy.

Induction of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astrocytes.

Abstract: The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease

A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA

Abstract: We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA-NLS peptide. The PNA-NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA-NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA-PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants

Abstract: Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.