Showing papers in "Nature Biotechnology in 1994"
TL;DR: The scaffold induced chondrocyte differentiation with respect to morphology and phenotype and represents a model cell culture substrate that may be useful for a variety of tissue engineering applications.
Abstract: Synthetic polymer scaffolds designed for cell transplantation were reproducibly made on a large scale and studied with respect to biocompatibility, structure and biodegradation rate. Polyglycolic acid (PGA) was extruded and oriented to form 13 μm diameter fibers with desired tenacity. Textile processing techniques were used to produce fibrous scaffolds with a porosity of 97% and sufficient structural integrity to maintain their dimensions when seeded with isolated cartilage cells (chondrocytes) and cultured in vitro at 37°C for 8 weeks. Cartilaginous tissue consisting of glycosaminoglycan and collagen was regenerated in the shape of the original PGA scaffold. The resulting cell-polymer constructs were the largest grown in vitro to date (1 cm diameter × 0.35 cm thick). Construct mass was accurately predicted by accounting for accumulation of tissue components and scaffold degradation. The scaffold induced chondrocyte differentiation with respect to morphology and phenotype and represents a model cell culture substrate that may be useful for a variety of tissue engineering applications.
1,224 citations
TL;DR: The flux balance methodology allows the quantitative interpretation of metabolic physiology, gives an interpretation of experimental data, provides a guide to metabolic engineering, enables optimal medium formulation, and provides a method for bioprocess optimization.
Abstract: Recently, there has been an increasing interest in stoichiometric analysis of metabolic flux distributions. Flux balance methods only require information about metabolic reaction stoichiometry, metabolic requirements for growth, and the measurement of a few strain–specific parameters. This information determines the domain of stoichiometrically allowable flux distributions that may be taken to define a strain's “metabolic genotype”. Within this domain a single flux distribution is sought based on assumed behavior, such as maximal growth rates. The optimal flux distributions are calculated using linear optimization and may be taken to represent the strain's “metabolic phenotype” under the particular conditions. This flux balance methodology allows the quantitative interpretation of metabolic physiology, gives an interpretation of experimental data, provides a guide to metabolic engineering, enables optimal medium formulation, and provides a method for bioprocess optimization. This spectrum of applications, and its ease of use, makes the metabolic flux balance model a potentially valuable approach for the design and optimization of bioprocesses.
1,006 citations
TL;DR: Evaluation of disease development in hybrids showed that combination of the two transgenes gave substantially greater protection against the fungal pathogen Cercospora nicotianae, causal agent of frogeye, than either transgene alone.
Abstract: Plants respond to pathogen attack by the induction of a battery of defenses, suggesting that different protective mechanisms may have complementary roles in the overall expression of disease resistance. We have investigated possible functional interactions between two different hydrolytic enzymes, chitinase and glucanase, by constitutive co-expression in transgenic tobacco of genes encoding the rice RCH10 basic chitinase and the alfalfa AGLU1 acidic glucanase. Hybrid plants were generated by crossing transgenic parental lines exhibiting strong constitutive expression of cauliflower mosaic virus (CaMV) 35S enhancer / RCH10 and CaMV 35S double promoter / AGLU1 gene fusions, respectively. Evaluation of disease development in these hybrids, heterozygous for each transgene, and in homozygous selfed progeny, showed that combination of the two transgenes gave substantially greater protection against the fungal pathogen Cercospora nicotianae, causal agent of frogeye, than either transgene alone. Productive interactions between chitinase and glucanase transgenes in vivo point to combinatorial expression of antimicrobial genes as an effective approach to engineering enhanced crop protection against fungal disease.
494 citations
TL;DR: The mechanisms that might be responsible for transgene inactivation are discussed and means of stabilizing gene expression in transgenic plants are examined.
Abstract: Desirable new phenotypes created by the introduction of foreign DNA into plants are frequently unstable following propagation, leading to a loss of the newly acquired traits. This genetic instability is due not to deletion or mutation of the introduced DNA but rather to the inactivation of the transgene. In this review, we discuss the mechanisms that might be responsible for transgene inactivation and examine means of stabilizing gene expression in transgenic plants.
475 citations
473 citations
TL;DR: A strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template, which contrasts with existing means of “humanizing” rodent monOClonal antibody in that the antibodies derived are completely human.
Abstract: We have developed a strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template. Thus the heavy chain of a rodent antibody (MAb32) directed against human tumor necrosis factor alpha (TNF alpha) was cloned and paired as a template chain with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage were selected by binding to the antigen. The selected human light chains were in turn paired with a repertoire of human heavy chains displayed on phage, and the phage selected again. The isolated phage displaying human antibody fragments binding to TNF alpha also bound to a peptide comprising the N-terminal region of TNF alpha as with MAb32. One of the human Fab fragments was recloned for expression as a glycosylated human antibody in mammalian cells: Binding to TNF alpha could be competed with MAb32 or with anti-serum to the peptide, indicating the same epitope. The human antibody was found to have a binding affinity (Kd = 15 nM) similar to MAb32 (Kd = 26 nM). The process contrasts with existing means of "humanizing" rodent monoclonal antibodies in that the antibodies derived are completely human.
371 citations
TL;DR: The levels of αAI protein in the pea seeds were as high as in bean seeds and the peas were resistant to the cowpea and Azuki bean weevils, suggesting this gene is a candidate for a genetic engineering approach that would make other legumes resistant to bruchid infestations.
Abstract: Infestations of stored legume seeds by bruchid beetles, such as the cowpea weevil and the Azuki bean weevil cause substantial economic and nutritional losses of these food crops, especially in developing countries. Seeds of the common bean are resistant to these bruchids largely because of the presence of α-amylase inhibitor (αAI-Pv), a seed protein that is toxic to the larvae. The αAI-Pv gene is therefore a candidate for a genetic engineering approach that would make other legumes (pea, chickpea, cowpea, Azuki bean) resistant to bruchid infestations. We tested this possibility by transforming peas (Pisum sativum) with the αAI-Pv gene driven by a strong seed-specific promoter. The levels of αAI protein in the pea seeds were as high as in bean seeds and the peas were resistant to the cowpea and Azuki bean weevils.
329 citations
TL;DR: In this article, the use of vectors containing the Tn903 kanr gene conferring G418-resistance was reported, which showed that copy number showed a tight correlation with drug resistance.
Abstract: Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.
328 citations
TL;DR: Results indicate that many wheat chromosomes contain homoeologous genes and genomk DNA fragments in a similar order to that found on rice chromosomes, which may provide a basis for novel gene isolation strategies in wheat.
Abstract: We have compared the structure of the genomes of rice and hexaploid bread wheat, which speciated more than 60 million years ago, by reciprocal mapping of DNA probes. Results indicate that many wheat chromosomes contain homoeologous genes and genomk DNA fragments in a similar order to that found on rice chromosomes. This conservation of gene order along chromosomes differing in DNA content by some 30–fold, may provide a basis for novel gene isolation strategies in wheat, one of the world's major food crop species.
278 citations
TL;DR: Though complex, apoptosis appears amenable to therapeutic intervention and offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.
Abstract: In a landmark paper published over two decades ago, Kerr et al. proposed the term apoptosis “for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations”1. In the ensuing years, this natural cell death process was studied at the basic science level, primarily with a view to understanding its roles in cancer and in the development and maintenance of the immune system. More recently, however, evidence has suggested a role for the failure of normal apoptosis control in many of the major diseases of the industrialized world. Though complex, apoptosis appears amenable to therapeutic intervention. The range of modern pharmaceutical strategies available to treat such disregulated gene-directed processes offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.
244 citations
TL;DR: Enhanced SOD activity in the mitochondria had only a minor effect on ozone tolerance, however, overproduction of SOD in the chloroplasts resulted in a 3–4 fold reduction of visible ozone injury.
Abstract: To evaluate the feasibility of using engineered antioxidant enzymes as an approach to improve the tolerance of plants to ambient stress, we have constructed transgenlc tobacco plants that overproduce superoxide dismutase (SOD), an enzyme which converts superoxide radicals into hydrogen peroxide and oxygen, and is believed to play a crucial role in antioxidant defense. We have targeted the MnSOD from Nicotiana plumbaginifolia either to the chloroplasts or to the mitochondria, and evaluated the ozone tolerance of transgenic and control plants. Enhanced SOD activity in the mitochondria had only a minor effect on ozone tolerance. However, overproduction of SOD in the chloroplasts resulted in a 3–4 fold reduction of visible ozone injury.
TL;DR: This review discusses issues and examines transformation systems recently developed to selectively eliminate particular transgene sequences from the final transgenic plant.
Abstract: The ability to efficiently introduce foreign genes into plants is key to the success of the emerging plant biotechnology industry. Genetic transformation of crop plants is becoming increasingly routine both in terms of the number of plant species which can be transformed and the frequency of transformation, resulting in a number of transgenk products which are ready or close to market introduction1. The imminent commercialization of transgenk plants has generated debate about the desirability of the transgenk products containing selectable marker genes, or in fact any other ancillary DNA sequences not directly contributing to the final product2–6. In this review, we discuss these issues and examine transformation systems recently developed to selectively eliminate particular transgene sequences from the final transgenic plant.
TL;DR: The freeze/thaw treatment can be utilized as a general method for the isolation of recombinant proteins from E. coli.
Abstract: Repeated cycles of freezing and thawing are sufficient to separate highly expressed recombinant proteins away from the cellular milieu of E. coli. Freezing and thawing liberates recombinant proteins from the bacterial cytoplasm, but does not release the bulk of endogenous E. coli proteins. Furthermore, protein secretion is not required. Fractionation of overexpressed proteins by freeze/thaw treatment does not depend on the identity of the recombinant protein and has been observed for thirty-five different recombinant proteins expressed in E. coli. These include proteins originally found in plant, animal or microbial sources, as well as several proteins designed de novo. Freezing and thawing typically yields approximately 50% of the recombinant protein in relatively pure form. Thus the freeze/thaw treatment can be utilized as a general method for the isolation of recombinant proteins from E. coli.
TL;DR: These G1 birds have reached sexual maturity and have been bred to produce transgenic offspring, demonstrating that stable transmission of foreign DNA can be obtained by the method developed.
Abstract: We have developed a method for production of transgenic chickens by DNA microinjection of chick zygotes followed by ex vivo embryo culture. The fate of plasmid DNA microinjected into the germinal disc of zygotes was analyzed in embryos which survived for at least 12 days in culture. Approximately half of the embryos contained plasmid DNA, 6% at a level equivalent to one copy per cell in all tissues analyzed. Seven chicks, 5.5% of the total number of injected ova, survived to sexual maturity. One of these, a cockerel, transmitted the exogenous DNA to 3.4% of his offspring. These G1 birds have reached sexual maturity and have been bred to produce transgenic offspring, demonstrating that stable transmission of foreign DNA can be obtained by our method.
TL;DR: Overexpression of protein disulfide isomerase from a single chromosomally integrated copy in Saccharomyces cerevisiae results in ten–fold higher levels of secretion of human platelet derived growth factor B homodimer, and a four–fold increase in secretion of Schizosaccharomyceces pombe acid phosphatase.
Abstract: Overexpression of protein disulfide isomerase (PDI) from a single chromosomally integrated copy in Saccharomyces cerevisiae results in ten-fold higher levels of secretion of human platelet derived growth factor B homodimer, and a four-fold increase in secretion of Schizosaccharomyces pombe acid phosphatase. This result provides evidence that inefficient protein folding limits the secretion of some heterologous proteins, and that manipulation of the endoplasmic reticulum lumenal environment can help overcome this limitation.
TL;DR: A rapid, convenient and efficient method of affinity purification which can overcome the rate-limiting step of the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein.
Abstract: In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient and efficient method of affinity purification which can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant 'fusion protease' cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.
TL;DR: A synthetic TAP–encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene was designed and assembled and fused it to a novel hybrid secretory prepro leader sequence, yielding biologically active rTAP.
Abstract: Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.
TL;DR: It is found that mutations in rpoH, the RNA polymerase sigma factor responsible for heat shock protein synthesis, affect the stability of heterologous secreted proteins.
Abstract: Even though secretion offers numerous advantages for the production of proteins in Escherichia coli, the expression of many heterologous proteins is severely limited by degradation in the periplasmic space. We found that mutations in rpoH, the RNA polymerase sigma factor responsible for heat shock protein synthesis, affect the stability of heterologous secreted proteins. A particularly dramatic increase in expression was further observed in rpoH degP double mutants. To minimize proteolytic degradation, we constructed a family of 25 isogenic strains deficient in all known cell envelope proteases (DegP, Protease III, Tsp(Prc), and OmpT), as well as the rpoH15 mutant allele, and characterized their growth in both shake flasks and fermentors. The availability of this set of strains permits the selection of a suitable host based on the optimal combination between the optimum reduction in protease activity and acceptable growth properties.
TL;DR: The tomato yellow leaf curl virus gene that encodes the capsid protein (VI) was placed under transcriptional control of the cauliflower mosaic virus 35S promoter and cloned into an Agrobacterium Ti-derived plasmid and used to transform plants from an interspecific tomato hybrid, sensitive to the TYLCV disease.
Abstract: The tomato yellow leaf curl virus (TYLCV) gene that encodes the capsid protein (V1) was placed under transcriptional control of the cauliflower mosaic virus 35S promoter and cloned into an Agrobacterium Ti-derived plasmid and used to transform plants from an interspecific tomato hybrid, Lycopersicon esculentum X L. pennellii (F1), sensitive to the TYLCV disease. When transgenic F1 plants, expressing the V1 gene, were inoculated with TYLCV using whiteflies fed on TYLCV-infected plants, they responded either as untransformed tomato or showed expression of delayed disease symptoms and recovery from the disease with increasingly more resistance upon repeated inoculation. Transformed plants that were as sensitive to inoculation as untransformed controls expressed the V1 gene at the RNA level only. All the transformed plants that recovered from disease expressed the TYLCV capsid protein.
TL;DR: It is shown that the first 102 bases of the viral capsid gene function as a translational enhancer, and that SFV vectors incorporating this RNA increase heterologous protein synthesis to the level of wild type polyprotein.
Abstract: We recently described a system for heterologous gene expression in a variety of mammalian cell types that is based on an efficiently replicating Semliki Forest virus (SFV) variant in which an RNA encoding a foreign protein replaces the RNA that normally encodes the viruses' structural polyprotein. Although expression levels are sufficiently high for many purposes, in general they are only 10% of the level of the polyprotein in a wild type SFV infection. Here we show that the first 102 bases of the viral capsid gene function as a translational enhancer, and that SFV vectors incorporating this RNA increase heterologous protein synthesis to the level of wild type polyprotein.
TL;DR: A flow–through device which uses high frequency, low energy ultrasonic resonance fields to transiently aggregate hybridoma cells and return them by sedimentation to a perfusion bioreactor retained up to 99 percent of the inflowing viable cells with no measurable effect on viability.
Abstract: We have developed a flow–through device which uses high frequency, low energy ultrasonic resonance fields to transiently aggregate hybridoma cells and return them by sedimentation to a perfusion bioreactor. The system retained up to 99 percent of the inflowing viable cells with no measurable effect on viability. Viable cells were selectively retained at up to 3 percent higher efficiency than nonviable cells. A stirred tank bioreactor was operated for 700 hours with acoustic cell recycle. Concentrations greater than 5×107 cells/ml were attained with a 5–fold increase in antibody concentration and a 70–fold increase in volumetric productivity compared with batch culture.
TL;DR: Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer to the development of scaled–up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations.
Abstract: We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells. Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors, detected as long-term culture-initiating cells (LTC-IC), could be achieved as well in stirred suspension cultures as in static cultures. Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment. Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC, CFC and total cell numbers in stirred cultures. Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold, respectively, above input values. Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer. Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations. In addition, stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms.
TL;DR: The white–flowering trait was maintained well through vegetative propagation; however, during growth of individual white-flowering plants, some pink color was found in some flowers.
Abstract: Chimeric chalcone synthase (CHS) constructs were prepared in both anti-sense and sense orientations, and introduced into the chrysanthemum cultivar Moneymaker, along with a T-DNA vector lacking a CHS construct. For both the anti-sense and sense constructs, the majority of the plants produced pink flowers typical of Moneymaker itself. Of 133 sense and 83 anti-sense transgenic individuals 3 of each set produced fully white or very pale pink flowers. No white-flowering transgenic plants were obtained in control transformations. The white flowers were found to accumulate higher levels of chalcone synthase precursors and to have reduced levels of chalcone synthase message. A small-scale field trial was performed to evaluate the stability of the phenotype throughout a series of vegetative propagation steps and during plant growth. The white-flowering trait was maintained well through vegetative propagation; however, during growth of individual white-flowering plants, some pink color was found in some flowers. At one site 2% of the white-flowering plants produced a few pink flowers; at two other sites, as many as 10-12% of the plants produced pale pink flowers.
TL;DR: The construction of a Saccharomyces cerevisiae strain expressing the gene encoding the L(+)–lactate dehydrogenase [L(+)-LDH)] from Lactobacillus casei is described, able to perform a mixed lactic acid–alcoholic fermenation.
Abstract: We describe the construction of a Saccharomyces cerevisiae strain expressing the gene encoding the L(+)–lactate dehydrogenase [L(+)–LDH)] from Lactobacillus casei. The recombinant strain is able to perform a mixed lactic acid–alcoholic fermenation. Yeast cells expressing the L(+)–LDH gene from the yeast alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid simultaneously convert glucose to both ethanol and lactate, with up to 20% of the glucose transformed into L(+)–lactate. Such strains may be used in every field where both biological acidification and alcoholic fermentation are required.
TL;DR: Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae, and a new protein with a broader activity was generated, suggesting its use as an alternative for CryIC in resistance management programs.
Abstract: To obtain Bacillus thuringiensis crystal proteins with new properties and to identify the regions involved in insecticidal activity, we generated hybrid genes composed of cryIC and cryIE by in vivo recombination. Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae. Transfer of this domain to CryIE, which is not active against these insects, resulted in a new protein with a broader activity. This hybrid protein binds to different receptors than CryIC, suggesting its use as an alternative for CryIC in resistance management programs.
TL;DR: It is shown that antisense inhibition of polyphenol oxidase (PPO) gene expression abolishes discoloration after bruising of potato tubers in individual transgenic lines grown under field conditions, opening the possibility of preventing enzymatic browning in a wide variety of food crops without resorting to treatments such as heating or the application of antioxidants.
Abstract: Spoilage caused by post–harvest enzymatic browning is a problem of considerable importance to food growers, processors and retailers. Here we show that antisense inhibition of polyphenol oxidase (PPO) gene expression abolishes discoloration after bruising of potato tubers in individual transgenic lines grown under field conditions. Using appropriate promoters to express antisense PPO RNA, melanin formation can be specifically inhibited in the potato tuber. This lack of bruising sensitivity in transgenic potatoes, and the absence of any apparent detrimental side effects open the possibility of preventing enzymatic browning in a wide variety of food crops without resorting to treatments such as heating or the application of antioxidants.
TL;DR: The utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity is demonstrated and antibody triggering yielded defined PCR product and higher sensitivity because of the absence of non-specific products.
Abstract: We demonstrate the utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity. One of the limitations of the polymerase chain reaction (PCR) is the co-amplification of non-specific products caused by TaqPol activity on low stringency templates present in the initial cycle of PCR. We have used anti-TaqPol antibodies as thermolabile switches that inhibit TaqPol activity at low temperatures (20-40 degrees C) and release fully active TaqPol when they are inactivated by elevated temperatures in the PCR thermal cycling (70-98 degrees C). Several in a set of high affinity anti-TaqPol monoclonal antibodies fully inhibited TaqPol activity at 37 degrees C. The capacity for inhibition was ablated by incubation at temperatures high enough to denature antibodies but not sufficiently high to significantly reduce TaqPol activity. In a PCR model system, preincubation of TaqPol with these antibodies yielded PCR product consisting entirely of the intended product and the absence or significant reduction of non-specific products and primer dimers. In evaluation of clinical samples such antibody triggering yielded defined PCR product and higher sensitivity because of the absence of non-specific products.
TL;DR: A second generation malaria transmission-blocking vaccine candidate based on Pfs25, the predominate surface protein of Plasmodium falciparum zygotes, appears to be a more potent antigen and immunogen than the original construct, and the fermentation and post-fermentation processing methodology easily lend themselves to technology transfer to the ultimate users, newly industrialized countries.
Abstract: We have constructed a second generation malaria transmission-blocking vaccine candidate based on Pfs25, the predominate surface protein of Plasmodium falciparum zygotes, to overcome potential production problems with the original construct. Four modifications were made: (1) addition of the last cysteine residue of the fourth epidermal growth factor like-domain of Pfs25; (2) mutagenesis of asparagine-linked glycosylation sites with glutamine rather than alanine; (3) addition of a six histidine tag at the carboxy-terminus for highly efficient purification of recombinant protein on nickel-NTA agarose; and (4) fermentation that combines continuous glucose fed-batch methodology with pH-controlled glucose addition and a terminal ethanol feed. The resulting product, TBV25H (Transmission-Blocking Vaccine based on Pfs25 with a Histidine tag), appears to be a more potent antigen and immunogen than the original construct, and the fermentation and post-fermentation processing methodology easily lend themselves to technology transfer to the ultimate users, newly industrialized countries.
TL;DR: An expanded bed adsorption procedure for efficient recovery of a recombinant fusion protein, directly from a crude fermentor broth without prior cell removal, is suitable for large–scale bioprocesses and allows efficient removal of cells, host proteins, contaminating DNA and endotoxins.
Abstract: We have used an expanded bed adsorption procedure for efficient recovery of a recombinant fusion protein, directly from a crude fermentor broth without prior cell removal. The fusion protein was designed to have a relatively low isoelectric point (pI) to allow anionic exchange adsorption at pH 5.5 where most Escherichia coli host proteins are not adsorbed. The gene product was secreted to the culture medium of the E. coli host cells in high yields (550 mg/l). The separation of cells and the concentration and recovery of the fusion protein could therefore be achieved by a single unit operation. The yield after the expanded bed adsorption exceeded 90 percent. Furthermore, the significant volume reduction by the expanded bed adsorption, enabled efficient and straight–forward polishing of the product by a subsequent affinity chromatography step, for removal of contaminating DNA and pyrogenic compounds to levels acceptable for regulatory authorities. An overall yield exceeding 90 percent was maintained after the affinity chromatography polishing step. The procedure outlined here is suitable for large–scale bioprocesses and allows efficient removal of cells, host proteins, contaminating DNA and endotoxins.