Showing papers in "Nature Biotechnology in 1993"

Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions

Abstract: We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.

Recent advances in the expression of foreign genes in Pichia pastoris

Abstract: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm

Abstract: We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.

Field Performance of Elite Transgenic Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis

Abstract: We introduced a synthetic gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis into immature embryos of an elite line of maize using microprojectile bombardment. This gene was expressed using either the CaMV 35S promoter or a combination of two tissue specific promoters derived from maize. High levels of CryIA(b) protein were obtained using both promoter configurations. Hybrid maize plants resulting from crosses of transgenic elite inbred plants with commercial inbred lines were evaluated for resistance to European corn borer under field conditions. Plants expressing high levels of the insecticidal protein exhibited excellent resistance to repeated heavy infestations of this pest.

Use of Peptide Libraries to Map the Substrate Specificity of a Peptide-Modifying Enzyme: A 13 Residue Consensus Peptide Specifies Biotinylation in Escherichia coli

Abstract: I describe a technique for screening peptide libraries of over 10(9) independent clones for substrates of peptide-modifying enzymes. The peptides, linked to their genetic material by the lac repressor, are exposed to the enzyme and then screened by affinity purification on a receptor specific for the modified product. The enzyme characterized, E. coli biotin holoenzyme synthetase, normally adds biotin to a specific lysine residue in complex protein domains. The 13 residue substrate identified by this library screening approach is much smaller than the 75 amino acid required sequence of the natural substrate, and can function at either end of a fusion protein. The sequence is quite distinct at some positions from that region of the natural substrates, presumably because the peptides have to mimic the folded structure formed by the natural substrate. This technique should be useful for mapping the substrate specificity of a variety of peptide-modifying enzymes. In addition, small peptide substrates that are enzymatically biotinylated at a single site should be useful for a variety of purposes in labeling, purification, detection, and immobilization of proteins.

Insect Resistant Rice Generated by Introduction of a Modified δ-endotoxin Gene of Bacillus thuringiensis

Abstract: As a first step towards development of insect resistant rice we have introduced a truncated δ-endotoxin gene, cryIA(b) of Bacillus thuringiensis (B.t.) which has specific biological activity against lepidopteran insects into a japonica rice. To highly express the cryIA(b) gene in rice the coding sequence was extensively modified based on the codon usage of rice genes. Transgenic plants efficiently expressed the modified cryIA(b) gene at both mRNA and protein levels. Bioassays using R2 generation plants with two major rice insect pests, striped stemborer (Chilo suppressalis) and leaffolder (Cnaphalocrosis medinalis), indicated that transgenic rice plants expressing the CrylA(b) protein are more resistant to these pests than untransformed control plants. Our results suggest that the B.t. endotoxin genes will be useful for the rational development of new rice varieties resistant to major insect pests.

Semliki Forest virus expression system: production of conditionally infectious recombinant particles.

Abstract: In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos

Abstract: We have developed an improved protocol for the rapid and efficient production of transgenic wheat. Three plasmids, each containing the selectable bar gene for resistance to the herbicide Basta and the β-glucuronidase (GUS) reporter gene, were delivered via particle bombardment, directly into immature embryos of two spring and one winter cultivar of wheat four days to two months after culture. Resistant calli were selected on phosphinothricin (PPT) media and screened for histochemical GUS activity. Twelve independent callus lines showing phosphinothricin acetyltransferase (PAT) activity were recovered from the bombardment of 544 explants (374 immature embryos and 170 one or two month old calli). R0 plants were regenerated from seven of these lines, of which so far five have produced R1 progeny, and two of the latter have produced R2 progeny. PAT activity was detected in each of the plants tested from the seven R0 lines, as well as in a 1:1 or 3:1 ratio in R1 plants following cross or self pollination, respectively. Resistance to topical application of Basta was seen in PAT positive plants and transgenic progeny. Molecular analysis by Southern hybridization showed the presence of the bar gene in all PAT positive R0 and R1 plants analyzed. Hybridization of the bar gene probe with high molecular weight DNA further confirmed integration into nuclear DNA. Both male and female transmission of the bar gene, and its segregation as a dominant Mendelian trait in R1 and R2 plants, were demonstrated. Flowering transgenic R0 plants could be obtained in 7–9 months following excision and culture of immature embryos.

Application of magnetic beads in bioassays.

Abstract: This review outlines the possibilities of magnetic separation techniques and the application of magnetic beads in bioassays. By linking monoclonal antibodies or DNA to magnetic beads, or by using magnetic beads coated with streptavidin, a specific interaction with the corresponding target is ensured. By means of an external magnet, the recovery of material for further studies is greatly simplified. Magnetic beads have proven valuable in cell separations, for example, removal of tumor cells from bone marrow and isolation of lymphoid cells from peripheral blood, and for the isolation, identification and genetic analysis of specific nucleic acid sequences (DNA or RNA) and for isolation of DNA binding proteins. In addition, some of these techniques have also proven to be useful in the detection of specific nucleic acids from viruses or bacteria.

Human antibody fragments specific for human blood group antigens from a phage display library.

Abstract: We have isolated human antibody fragments with binding specificities against the blood group antigens of the ABO and I blood group systems (B and HI), of the Rh system (D and E) and of the Kell system (Kpb), by selecting a phage-antibody library on human red cells. The fragments, expressed in bacteria, were antigen-specific and effective in assays including agglutination and immunohistochemistry. Selection of phage-antibody libraries using intact cells seems to offer a promising alternative to hybridoma technology for the production of antibodies against cell surface molecules.

In situ product removal as a tool for bioprocessing.

Abstract: In situ product removal (ISPR) is the fast removal of product from a producing cell thereby preventing its subsequent interference with cellular or medium components. Over the past 10 years ISPR techniques have developed substantially and its feasibility (with improvements in yield or productivity) for several processes demonstrated. Assessment of progress, however, compared to the potential benefits inherent in the ISPR approach to bioprocessing reveals that these are far from being exploited fully. Here we indicate future directions including application of the ISPR approach to a wider range of product groups and the development of novel, more specific ISPR methodologies, applicable under sterile conditions in the immediate vicinity of the producing cells. General guidelines for adaptation of an appropriate ISPR approach for a given product are also provided.

Phytase-containing Transgenic Seeds as a Novel Feed Additive for Improved Phosphorus Utilization

Abstract: Phytate is the main storage form of phosphorus in many plant seeds, but bound in this form it is a poor nutrient for monogastric animals Its availability can be significantly increased by addition of the enzyme phytase, which releases phosphate from the substrate, phytate We have engineered a phytase from Aspergillus niger in tobacco seeds, providing a stable and convenient packaging of the enzyme that is directly applicable in animal feed The enzyme was expressed as 1 percent of the soluble protein in mature seeds In in vitro tests that simulated chicken crop and stomach conditions, release of phosphate from feed by addition of transgenic seeds was demonstrated Supplementation of broiler diets with transgenic seeds resulted in an unproved growth rate, comparable to diets supplemented with fungal phytase or phosphorus Addition of phytase-transgenic seeds to animal feed thus obviates the need for inorganic phosphorus supplementation, and is environmentally desirable because of the reduced excretion of phosphorus

Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli.

Abstract: The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form "miniantibodies" with significantly higher avidity than in the case of leucine zipper containing constructs The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment We report here a high-cell density fermentation of E coli producing these miniantibodies and a work-up procedure suitable for large scale production Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E coli culture

Stable Transformation of Picea glauca by Particle Acceleration

Abstract: By defining the somatic embryo developmental stage which expressed β-glucuronidase (GUS) at a high level yet was also competent to form embryogenic callus at a high frequency under selection, we obtained transformed Picea glauca (white spruce) embryogenic callus, embryos and seedlings expressing GUS in all cells. Plasmid DNA, containing three chimeric constructs [enhanced cauliflower mosaic virus (CaMV) 35s-GUS, nopaline synthase-neomycin phosphotransferase (NPTII), and CaMV 35s-Bacillus thuringiensis (B.t.) cryIA endotoxin] was introduced into four developmental stages of white spruce somatic embryos by particle acceleration. Transient expression was observed in all of the stages of somatic embryos tested, but transformed embryogenic callus was induced only from the two most advanced. Embryogenic callus was identified by histochemical staining for GUS as early as 6 weeks following particle acceleration. All GUS positive embryogenic callus lines also showed NPTII activity. Incorporation of the introduced genes into the genome was confirmed by PCR and Southern blot analysis of embryogenic callus and regenerated transformed plants. Plants derived from several transformed embryogenic callus lines are currently undergoing acclimatization in the greenhouse. Spruce budworm (Chorisoneura fumiferana) feeding trials with embryogenic callus and transformed seedlings indicate a low, sublethal level of B.t. expression.

Culture pH Affects Expression Rates and Glycosylation of Recombinant Mouse Placental Lactogen Proteins by Chinese Hamster Ovary (CHO) Cells

Abstract: Glycosylation patterns and specific expression rates of the recombinant protein mouse placental lactogen-I (mPL-I) by Chinese hamster ovary (CHO) cells varied significantly over the extracellular pH (pHe) range of 6.1 to 8.7. The maximum specific mPL-I expression rates occurred between pHe 7.6 and 8.0. The pHe effect on protein expression was confirmed using a different CHO cell expressing the unglycosylated recombinant protein mouse placental lactogen-II (mPL-II). Decreases in the extent of glycosylation of mPL-I were observed at low (below 6.9) and high (above 8.2) pHe values. The pHe dependent variations in mPL-I accumulation in the supernatant as well as in glycosylation patterns were not the result of enzymatic degradation in the culture medium.

Transformation of Elite Cotton Cultivars via Particle Bombardment of Meristems

Abstract: A variety-independent protocol was developed to allow the production of transgenic cotton. High velocity gold beads coated with DNA were used to deliver foreign genes directly into the meristematic tissue of excised embryonic axes. Bombarded explants were allowed to develop into plants which were subsequently screened for gus gene activity. Buds in the axils of transformed leaves were forced to develop into plants by pruning away non-transformed primary shoot tips. Plants derived from this process carried the foreign gene in one or more of their tissue layers. Transformation frequencies varied with the genotype used, but all cultivars attempted to date have yielded transgenic progeny. Molecular and genetic characterization of primary transformants and their progeny established that foreign genes were stably integrated and transmitted to progeny in a Mendelian fashion.

Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator.

Abstract: One link in the complex chain of medical economics is the cost of bringing new drugs and biologicals to the market. Advances in recombinant-DNA technology permit production of therapeutically active proteins in effectively unlimited quantities. Nevertheless, each expression system has a characteristic influence on the nature of the product produced and the process required to obtain it. In this case study we compare experiences with recombinant-tissue plasminogen activator (rtPA) produced in Chinese hamster ovary (CHO) cells and in Escherichia coli, with the aim of understanding the roles of some of the parameters that affect process economics. tPA belongs to the group of highly specific serine proteases that convert plasminogen to plasmin, which in turn degrades several protein substrates including fibrin, thus making it an effective thrombolytic agent. The treatment of acute myocardial infarction with such thrombolytic agents can result in early discharge of patients and decreased medical costs. However, there are major differences in the prices of the various available agents. The price of the FDA-licensed tPA product is $2,200 per dose or $22,000 per gram. It is believed that a significant portion of this price relates to manufacturing costs. We examine by way of case study illustration the cost breakdown for the two processes, and highlight important process, design and economic considerations that ultimately define a particular protein product.

Stable Plastid Transformation in PEG-treated Protoplasts of Nicotiana tabacum

Abstract: Plastid engineering currently relies on DNA delivery by the biolistic process. We report here stable plastid transformation in tobacco by an alternate direct transformation protocol that is based on polyethylene glycol (PEG) treatment of leaf protoplasts in the presence of the transforming DNA. Clones with transformed plastid genomes were selected by spectinomycin resistance encoded by a mutant 16S ribosomal RNA gene. Incorporation of the transforming DNA into the plastid genome was confirmed by two unselected markers, streptomycin resistance and a novel PstI site that flank the spectinomycin resistance mutation in plasmid pZS148. Our simple and inexpensive protocol eliminates the dependence on the particle gun for chloroplast transformation and should facilitate applications of plastome engineering in crops.

A New Tobacco Mosaic Virus Vector and its Use for the Systemic Production of Angiotensin-I-Converting Enzyme Inhibitor in Transgenic Tobacco and Tomato

Abstract: We have developed a new tobacco mosaic virus (TMV) RNA vector and have used it initially to systemically produce an angiotensin-I-converting enzyme inhibitor peptide (ACEI) in tobacco and tomato plants. This vector incorporates a six base 3′ context sequence, which permits readthrough of the stop codon for the TMV 130K protein gene, inserted between the stop codon for the coat protein (CP) gene and ACEI gene. In contrast to previous TMV RNA vectors, the new vector produced both an intact CP and a fused protein consisting of CP and ACEI (CP-ACEI). As a result, the vector could form virus particles and spread systemically from inoculated to non-inoculated leaves. In tomato plants, production of ACEI in fruit was also achieved.

Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli.

Abstract: The efficient production of the textile dye indigo by fermentation has been a goal since the early 1980's when the first bacterial strains capable of this synthesis were constructed. We report here the development of a recombinant microorganism that directly synthesizes indigo from glucose. This construction involved the cloning and genetic manipulation of at least 9 genes and modifications of the fermentation medium to help stabilize the biosynthetic activity. Directed genetic changes in two operons caused significant increases in reaction rates and in the stability of the catalytic enzymes. This example of whole cell catalysis by a recombinant Escherichia coli represents a novel and environmentally sound approach to the synthesis of a high value specialty chemical.

Transposition Mediated Re–positioning and Subsequent Elimination of Marker Genes from Transgenic Tomato

Abstract: Transposition Mediated Re–positioning and Subsequent Elimination of Marker Genes from Transgenic Tomato

Safety assessment of the neomycin phosphotransferase II (NPTII) protein.

Abstract: Two approaches were used to assess the safety of the NPTII protein for human consumption using purified E. coli produced NPTII protein that was shown to be chemically and functionally equivalent to the NPTII protein produced in genetically engineered cotton seed, potato tubers and tomato fruit. The NPTII protein was shown, as expected, to degrade rapidly under simulated mammalian digestive conditions. An acute mouse gavage study confirmed that the NPTII protein caused no deleterious effects when administered by gavage at a cumulative target dosage of up to 5000 mg/kg of body weight. This dosage correlates to at least a million fold safety factor relative to the average daily consumption of potato or tomato, assuming all the potatoes or tomatoes consumed contained the NPTII protein. These results, along with previously published information, confirm that ingestion of genetically engineered plants expressing the NPTII protein poses no safety concerns.

Agrobacterium -Mediated Transfer of Antisense Chalcone Synthase cDNA to Gerbera hybrida Inhibits Flower Pigmentation

Abstract: We have developed a regeneration method and an Agrobacterium-mediated transformation protocol for the commercially important ornamental plant Gerbera hybrida. Pieces of petioles of the red variety Terra Regina were cocultivated with a disarmed Agrobacterium tumefaciens vector containing a nearly full-length antisense cDNA encoding gerbera chalcone synthase under the control of CaMV 35S promoter, and a nos-nptII marker gene. The transformed cells were selected on the basis of kanamycin resistance and regenerated into flowering plants. Introduction of the antisense cDNA blocked anthocyanin synthesis and resulted in a dramatically altered flower pigmentation in some of the transformants.

Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei

Abstract: We have engineered the filamentous fungus Trichoderma reesei to assemble and secrete immunologically authentic engineered Fab antibody fragments into the culture medium. A major improvement in yield was achieved by fusing the heavy Fd chain to the T. reesei cellulase, CBHI. The yields of secreted, immunologically active Fab and CBHI-Fab fusion were 1 mg/1 and 150 mg/1, respectively. The Fab fragment can be released from the fusion protein CBHI-Fab by an extracellular T. reesei protease. There was no detectable difference in affinity for the antigen between the engineered Fab and the idiotypic antibody.

Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system.

Abstract: We describe here a continuous perfusion bioreactor system that enables a population of unselected human mononuclear bone marrow cells obtained from adult donors to expand up to 20 to 25-fold over a two-week period. Colony-forming units of granulocyte-macrophage (CFU-GM) progenitor cells expand 10 to 30-fold. These expansions depend on the gas phase oxygen concentration, the seeding density and time of cell harvest. Under operating conditions that allow for good cell proliferation, 3 to 4 million mononuclear cells can be obtained per square centimeter, with 0.5 to 0.8% being progenitor cells. Autologous human sera supported cell expansion as efficiently as animal sera. Increasing the size of the perfusion system to produce a clinically meaningful number of CFU-GMs could have important applications in bone marrow transplantation therapies.

The Effect of Folding Catalysts on the In Vivo Folding Process of Different Antibody Fragments Expressed in Escherichia coli

Abstract: The Fv and Fab fragment and both orientations of the single-chain Fv fragment (VH-linker-VL and VL-linker-VH) of an antibody can be expressed in functional form in the periplasm of Escherichia coli, but the yield of these correctly assembled proteins is limited by the periplasmic folding process. While the periplasmic E. coli disulfide isomerase DsbA is required for this assembly, its functional over-expression does not significantly change the folding limit. Similarly, the functionally over-expressed E. coli proline cis-trans isomerase does not change the amount of all but one of the antibody fragments, not even if DsbA is over-expressed as well. Therefore, aggregation steps in the periplasm appear to compete with periplasmic folding, and they may occur before disulfide formation and/or proline cis-trans isomerization takes place and be independent of their extent.

Molecular Analysis of Small Grain Cereal Genomes: Current Status and Prospects

Abstract: Recent developments in cereal genome analysis include generation of RFLP maps, flow sorting of chromosomes, identification of landmarks for genes and a more advanced model for cereal genome organization. These developments are reviewed together with new prospects for the isolation of defined genes from large cereal genomes and for the production of a composite map of the ancestral grass genome to aid in the genetic analysis of all the Gramineae. The advances that can now come from comparative genome mapping are likely to promote further the new era of plant genetics.

Expansion of Primitive Human Hematopoietic Progenitors in a Perfusion Bioreactor System with IL-3, IL-6, and Stem Cell Factor

Abstract: Expansion of Primitive Human Hematopoietic Progenitors in a Perfusion Bioreactor System with IL-3, IL-6, and Stem Cell Factor

Forestry in the 21st Century

Abstract: The biotechnology of somatic embryogenesis

Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains.

Abstract: The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed in Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface. Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays. In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added in the extracellular fluid. Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers. This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials. In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria.