Showing papers in "Nature Biotechnology in 1989"
TL;DR: RFLP linkage maps provide a more direct method for selecting desirable genes via their linkage to easily detectable RFLP markers and may make it possible to clone genes whose products are unknown, such as genes for disease resistance or stress tolerance.
Abstract: Breeders have traditionally improved plant varieties by selecting on the basis of phenotype. Now restriction fragment length polymorphism (RFLP) linkage maps are being constructed for most major crop plants and these maps provide a more direct method for selecting desirable genes via their linkage to easily detectable RFLP markers. The integration of RFLP techniques into plant breeding promises to: (1) Expedite the movement of desirable genes among varieties, (2) Allow the transfer of novel genes from related wild species, (3) Make possible the analysis of complex polygenic characters as ensembles of single Mendelian factors, and (4) Establish genetic relationships between sexually incompatible crop plants. In the future, high density RFLP maps may also make it possible to clone genes whose products are unknown, such as genes for disease resistance or stress tolerance.
1,239 citations
TL;DR: This review summarizes what is known about why IBs form and ways of increasing the production of soluble protein in bacterial systems and discusses possibilities for mimicking these mechanisms in bacteria via secretion, cloning of mammalian foldases, and mutation of the post-translational modification systems of the host bacteria.
Abstract: Production of recombinant proteins in bacteria is limited by the formation of cytoplasmic aggregates (inclusion bodies or “IBs”). This review summarizes what is known about why IBs form and ways of increasing the production of soluble protein in bacterial systems. The easiest way to lower IB formation is to reduce the growth temperature of the bacteria. IB formation is not directly correlatable with the production rate, nor with the size of the produced protein. The primary sequences of a few proteins that do not form IBs at higher production temperatures contain either a low content of proline residues or stretches of acidic amino acids. Metal ion binding may also lower the tendency to form IBs at growth temperatures above 30°C. Three aspects of protein synthesis in mammalian cells, compartmentation, interprotein interactions (sortases, foldases, unfoldases, and chaperonins), and post-translational modifications, have significant effects on the solubility of the proteins produced. Possibilities for mimicking these mechanisms in bacteria via secretion, cloning of mammalian foldases, and mutation of the post-translational modification systems of the host bacteria are discussed.
509 citations
TL;DR: Studies of both the refolding of denatured proteins in vitro, and of in vivo folding and maturation pathways, indicate that aggregates derive from specific partially folded intermediates and not from mature native, or fully unfolded proteins.
Abstract: The accumulation of newly synthesized polypeptide chains expressed from cloned genes as non native aggregates has become an important factor in the recovery of such proteins. Studies of both the refolding of denatured proteins in vitro, and of in vivo folding and maturation pathways, indicate that aggregates derive from specific partially folded intermediates and not from mature native, or fully unfolded proteins. The aggregation process in both homologous and heterologous cytoplasms may be driven by partial intracellular denaturation of intermediates, for example by high temperature, or by the absence of a critical factor—prosthetic group, sub-unit, chaperone—during the maturation process. All of these processes appear to be highly specific and subject to modification by genetic engineering of the intermediates, or alteration of their environment. This requires appreciation of the properties of such intermediates as distinct from the native states.
453 citations
TL;DR: Somatic embryogenesis represents a significant step in developing a new breeding strategy for apomictic banana and plantain species and histological examination confirmed bipolar organization of somatic embryos.
Abstract: Proembryogenic calli were initiated from basal leaf sheaths and rhizome tissue on modified Schenk and Hildebrandt (SH) medium with 30 μM 3,6–dichloro–2–meth–oxybenzoic acid (Dicamba). Cell suspensions were maintained in half–strength Murashige and Skoog (MS) medium supplemented with 20 μM Dicamba. The development of somatic embryos was promoted in cell suspensions 3–4 weeks after subculture in liquid modified MS medium with 5 μM zeatin. Characteristic stages of embryonic development were recapitulated and histological examination confirmed bipolar organization of somatic embryos. Conversion into plantlets took place in double layer media system composed of solid half strength MS medium with 5 μM zeatin and 1 g/l charcoal and liquid, hormone–free, half strength MS medium. In four Musa genotypes several hundred plantlets were regenerated and transferred into soil where they continued to grow. Somatic embryogenesis represents a significant step in developing a new breeding strategy for apomictic banana and plantain species.
220 citations
TL;DR: This paper obtained friable, non-mucilaginous embryogenic callus from cultured immature embryos of an elite maize inbred line derived from Iowa stiff stalk synthetic germplasm.
Abstract: We have obtained friable, non-mucilaginous embryogenic callus from cultured immature embryos of an elite maize inbred line derived from Iowa stiff stalk synthetic germplasm. This callus was used to generate embryogenic cell suspensions containing compact clumps of densely cytoplasmic cells. Plants were readily regenerated from the suspensions, which were also an excellent source of protoplasts. The suspension cultures maintained their plant regeneration capacity through many months. The cultures were cryopreserved in liquid nitrogen and embryogenic callus and suspension cultures were routinely recovered from cryopreserved cells. Protoplasts were isolated in large numbers from both primary and cryopreserved suspension cultures. Cultured protoplasts divided and formed embryogenic callus both when plated in agarose and on nurse cultures. Hundreds of plants were regenerated from protoplast-derived calli. Plants with viable progeny were obtained although many of the regenerates showed morphological and reproductive abnormalities.
219 citations
TL;DR: A hybrid gene to direct the synthesis of human anti-hemophilic factor IX to the mammary gland is designed and introduced into sheep, and two transgenic ewes, each carrying about 10 copies of the foreign gene, have been analysed for expression.
Abstract: Transgenic livestock may prove useful for the large scale production of valuable proteins. By targeting expression to the mammary gland these proteins could be harvested from milk. To this end, we have designed a hybrid gene to direct the synthesis of human anti-hemophilic factor IX to the mammary gland, and introduced it into sheep. Two transgenic ewes, each carrying about 10 copies of the foreign gene, have been analysed for expression. Both animals express human factor IX RNA in the mammary gland and secrete the corresponding protein into their milk.
193 citations
TL;DR: A portion of an Arabidopsis thaliana 2S albumin gene was replaced by sequences encoding the neuropeptide Leu-enkephalin, flanked by tryptic cleavage sites, to produce large amounts of biologically active peptides as part of chimeric plant seed storage proteins, the2S albumins.
Abstract: We have explored the possibility of producing large amounts of biologically active peptides as part of chimeric plant seed storage proteins, the 2S albumins. A portion of an Arabidopsis thaliana 2S albumin gene, encoding a region of the protein whose sequence is not highly conserved among different species, was replaced by sequences encoding the neuropeptide Leu-enkephalin, flanked by tryptic cleavage sites. Using Agrobacterium mediated transformation, the modified gene was transformed into Arabidopsis and oilseed rape and the 2S albumins isolated from the seeds of the regenerated transgenic plants. These were digested with trypsin and the enkephalin containing peptide isolated and treated to remove the extra lysine residue. Up to 200 nmol of peptide were recovered per gram of seed.
184 citations
TL;DR: The expression of a bovine chymosin cDNA in Trichoderma reesei was studied to test the ability of this novel fungal host vector system to express and secrete heterologous gene products.
Abstract: We have studied the expression of a bovine chymosin cDNA in Trichoderma reesei to test the ability of this novel fungal host vector system to express and secrete heterologous gene products. Four different expression plasmids were constructed to determine the optimum manner to fuse the chymosin cDNA to the promoter and the terminator of the major T. reesei cellulase gene, cellobiohydrolase I (cbh1). All four constructions, when transformed into Trichoderma, determined the secretion of a polypeptide corresponding in size to prochymosin and reacting with antichymosin antiserum. This polypeptide had a molecular weight indistinguishable from chymosin and showed milk clotting activity. In preliminary fermentation studies, one transformant secreted as much as 40 mg/l of active chymosin.
183 citations
TL;DR: This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity, and facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies.
Abstract: We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies. (Less)
175 citations
TL;DR: Results demonstrate that transgenic tomato plants containing a gene encoding a B.t. kurstaki HD-1 protein can provide control of lepidopteran insect pests under field conditions.
Abstract: Transgenic tomato plants expressing a lepidopteran-specific insect control protein from Bacillus thuringiensis var. kurstaki HD-1 (B.t.k.) were field tested in 1987 and 1988 to evaluate their efficacy under normal field conditions. When infested with eggs of the tobacco hornworm (Manduca sexta), the transgenic plants showed only very limited feeding damage on the leaves near the inoculated sites soon after the larvae hatched, and sustained no further damage. In contrast, nontransgenic control plants suffered heavy feeding damage and were almost completely defoliated within two weeks. Significant control of tomato fruitworm (Heliothis zea) and tomato pinworm (Keiferia lycopersicella) was also observed. These results demonstrate that transgenic tomato plants containing a gene encoding a B.t.k. protein can provide control of lepidopteran insect pests under field conditions.
174 citations
TL;DR: Analysis of herbicide resistant transgenic tobacco and potato lines resulted from Agrobacterium mediated transformation with chimeric bar genes encoding phosphinothricin acetyl transferase confirms that glufosinate can be applied as a selective post–emergence herbicide on engineered crops.
Abstract: We have analysed two herbicide resistant transgenic tobacco (Nicotiana tabacum) and four potato (Solanum tuberosum) lines under field conditions. These lines resulted from Agrobacterium mediated transformation of tobacco and potato leaf discs with chimeric bar genes encoding phosphinothricin acetyl transferase (PAT). Greenhouse tests have demonstrated that this enzyme protects transformed plants against the broad–spectrum herbicides glufosinate and bialaphos. In field tests, the transformants revealed the same agronomic performance as untransformed controls. Complete resistance to field dose applications of glufosinate was observed, although PAT expression in these lines varied by two orders of magnitude. These field data confirm that glufosinate can be applied as a selective post–emergence herbicide on engineered crops.
TL;DR: To expand the understanding of the breadth of protection conferred by CP genes, transgenic tobacco plants were produced that express a gene encoding the CP of the potyvirus soybean mosaic virus (SMV), a non-pathogen on tobacco.
Abstract: Protection Against Potyvirus Infection in Transgenic Plants: Evidence for Broad Spectrum Resistance
TL;DR: A strain of Pseudomonas mendocina is identified that oxidizes trichloroethylene (TCE) after growth in the presence of toluene and a DNA fragment from this organism, when introduced into the appropriate expression vector system, conferred upon Escherichia coli the ability to oxidize both toLUene and TCE.
Abstract: We have identified a strain of Pseudomonas mendocina that oxidizes trichloroethylene (TCE) after growth in the presence of toluene. A DNA fragment from this organism, when introduced into the appropriate expression vector system, conferred upon Escherichia coli the ability to oxidize both toluene and TCE. The recombinant E. coli rapidly degrades TCE to carbon dioxide, chloride ion and simple water soluble metabolites and reduces the concentration of TCE in water as much as 1000–fold. The extensive degradation of TCE by the recombinant E. coli is due to genetic manipulations that uncouple the regulation of TCE degrading enzymes from growth and co metabolism of toluene.
TL;DR: Results and preliminary data on morphological characteristics indicate that potato cultivars can be genetically engineered to contain new desirable traits with preservation of their intrinsic properties.
Abstract: We efficiently transformed the commercial cultivars of potato (Solanum tuberosum) Bintje, Desiree and Escort after optimizing the conditions for regeneration from potato tuber discs. For transformation, tuber discs were cocultivated with Agrobacterium tumefaciens using a disarmed binary vector system. This system allowed the introduction of a chimaeric gene encoding the coat protein (CP) of potato virus X (PVX) into two cultivars most susceptible to this virus, Bintje and Escort. Five transgenic plant lines with expression levels of CP higher than 0.1% of soluble leaf protein were analyzed for resistance to a challenging inoculation with PVX (1 μg/ml). We observed a delay in symptom development as well as a drastic reduction in the accumulation of virus. Furthermore, we found a correlation between the expression level of the CP–gene and the reduction in virus accumulation. Cytogenetic analysis of 62 independently obtained transgenic lines showed the normal tetraploid number of chromosomes (2n = 4x = 48) in 97 percent of the examined plants. Phenotypically all these plants appeared normal. One plant line exhibited an abnormal phenotype and contained about the octaploid number of chromosomes (4n ≈ 8x = 96). These results and preliminary data on morphological characteristics, as these are determined in the official variety registration procedure, indicate that potato cultivars can be genetically engineered to contain new desirable traits with preservation of their intrinsic properties.
TL;DR: Plant regeneration from maize (Zea mays L.) protoplasts with recovery of fertile plants from a Cateto inbred line adapted to tropical regions is reported and it is demonstrated that high plating efficiencies and plant regeneration from corn protoplast can be achieved with different culture methods.
Abstract: We report plant regeneration from maize (Zea mays L.) protoplasts with recovery of fertile plants from a Cateto inbred line adapted to tropical regions. Protoplasts were isolated from embryogenic cell suspension cultures capable of regenerating fertile plants. Cell colonies and embryogenic calli were obtained at high frequencies when protoplasts were cultured either in a thin layer of liquid medium or on cellulose-nitrate filters placed on a feeder layer of maize cells. High plating efficiencies were obtained with both culture methods using either a modified KM-8p or a modified N6 culture media. The results confirmed that maize protoplasts maintain the regeneration capacity of donor cells. We also demonstrated that high plating efficiencies and plant regeneration from maize protoplasts can be achieved with different culture methods. Furthermore, we recovered fertile plants with viable progenies from maize protoplasts.
TL;DR: A recombinant DNA-modified strain of the filamentous fungus Cephalosporium acremonium, strain LU4-79-6, produced more of the antibiotic cephalOSporin C than a non-recombinant strain, strain 394-4, from which it was derived by transformation.
Abstract: A recombinant DNA-modified strain of the filamentous fungus Cephalosporium acremonium, strain LU4-79-6, produced more of the antibiotic cephalosporin C than a non-recombinant strain, strain 394-4, from which it was derived by transformation. Strain 394-4, derived from C. acremonium ATCC 11550 by multiple rounds of mutagenesis and screening for improved antibiotic biosynthesis, has been useful for producing cephalosporin C at industrial scale. Strain LU4-79-6 has one insert of pPS56 DNA in its high molecular weight DNA. Plasmid pPS56 includes a dominant hygromycin B resistance marker and a 7 kb BamH1 C. acremonium DNA fragment containing the cefEF gene. The cefEF gene codes for a bifunctional protein that exhibits two sequentially-acting cephalosporin biosynthetic enzyme activities: deacetoxycephalosporin C synthetase (DAOCS) and deacetylcephalosporin C synthetase (DACS). Extracts of strain LU4-79-6 contained ∼2-fold more DAOCS activity than corresponding extracts of its non-recombinant parent. Strain LU4-79-6 excretes less penicillin N, the substrate of DAOCS, than strain 394-4. Resistance to hygromycin B, presence of pPS56 DNA, elevated intracellular DAOCS, decreased penicillin N production, and increased cephalosporin C production are retained after growth in a medium free of hygromycin B. The superiority of the recombinant strain has been confirmed at pilot scale.
TL;DR: The recombinant product is a single biologically active species that constitutes >80% of the protein in media from expressing cells; the signal sequence of the pre–lysozyme is accurately processed.
Abstract: We have cloned a cDNA for bovine lysozyme c2, a novel lysozyme characteristic of the cow stomach and expressed its protein product by secretion using its native signal sequence in the methylotrophic yeast, Pichia pastoris. A semi–continuous fermentation process used for production resulted in extremely high cell densities and product concentrations. During fed–batch fermentation, bovine lysozyme concentrations exceeded 550 mg/liter, and cell densities exceeded 120 g dry cells/liter. In continuous culture, bovine lysozyme and cell concentrations remained constant at approximately 350 mg/liter and 100 g dry cells/liter, respectively. The recombinant product is a single biologically active species that constitutes >80% of the protein in media from expressing cells; the signal sequence of the pre–lysozyme is accurately processed.
TL;DR: Animal studies have shown that exogenously added growth factors can accelerate the normal healing process, and these factors have also been used successfully in humans to treat previously incurable wounds.
Abstract: The process of wound healing begins immediately following surface lesions or when skin proteins become exposed to radiation, chemical damage or extreme temperatures. Wound repair requires close control of degradative and regenerative processes, involving numerous cell types and complex interactions between multiple biochemical cascades. Growth factors released in the traumatized area promote cell migration into the wound area (chemotaxis), stimulate the growth of epithelial cells and fibroblasts (mitogenesis), initiate the formulation of new blood vessels (angiogenesis), and stimulate matrix formation and remodeling of the affected region. Animal studies have shown that exogenously added growth factors can accelerate the normal healing process. Growth factors have also been used successfully in humans to treat previously incurable wounds. The most intensively studied growth factors are EGF, FGFs, PDGF, TGF-α, and TGF-βs. Each of these factors is currently the focus of intense commercial development.
TL;DR: Recombinant human macrophage colony-stimulating factor, a disulfide-linked dimeric protein containing 18 cysteines, has been purified in monomeric form from E. coli and renatured to generate fully active dimers with an overall yield of 25 percent.
Abstract: Recombinant human macrophage colony-stimulating factor (M-CSF), a disulfide-linked dimeric protein containing 18 cysteines, has been purified in monomeric form from E coli and renatured to generate fully active dimers with an overall yield of 25 percent M-CSF monomers were quantitatively refolded at concentrations as high as 07 mg/ml Residual contaminants, including endotoxin, were removed from the refolded M-CSF by hydrophobic interaction chromatography The kinetics of refolding and the characteristics of the renatured product were determined by measuring (1) molecular size, (2) biological activity, and (3) reactivity with a novel neutralizing monoclonal antibody specific for the dimeric form of M-CSF The results show that refolded M-CSF from E coli closely resembles both native and recombinant M-CSF produced by mammalian cells
TL;DR: Understanding of how bacteria integrate diverse external and internal stimuli into a concerted cellular response may be the key to successfully engineer commercially important microbes, and treat medically significant bacteria.
Abstract: The analysis of gene regulation in bacteria is entering a new phase. Global regulatory networks, signal processing, responses of complexly regulated sets of genes, and interactions between regulatory systems are becoming a focus of intensive research efforts. Our understanding of how bacteria integrate diverse external and internal stimuli into a concerted cellular response may be the key to successfully engineer commercially important microbes, and treat medically significant bacteria. Research on Pseudomonas has contributed to the overall progress in this field.
TL;DR: A gene from the soil bacterium Alcaligenes eutrophus JMP134 is used to introduce a herbicide degrading mechanism into plants and regenerated plants showed resistance when sprayed with the herbicide.
Abstract: We have used a gene from the soil bacterium Alcaligenes eutrophus JMP134 to introduce a herbicide degrading mechanism into plants. The gene, tfdA, which encodes a 2,4-dichlorophenoxyacetate monooxygenase (DPAM) catalyzing the first step in the bacterial 2,4-D degradative pathway, has recently been cloned and sequenced. The coding sequence, fused with plant expression sequences was introduced into plants via Agrobacterium-mediated gene transfer. The chimeric gene was expressed in tobacco (Nicotiana tabacum) under the control of either a constitutive or a light-inducible promoter. Transgenic tobacco lines tolerated elevated levels of 2,4-D in tissue culture, and regenerated plants showed resistance when sprayed with the herbicide.
TL;DR: The nucleotide sequence of the gene from Pseudomonas diminuta MG encoding parathion hydrolase was determined, and a single open reading frame located, and the deduced N-terminal amino acid sequence was compared with that determined by protein sequence analysis of the enzyme from P. diminuta as discussed by the authors.
Abstract: The nucleotide sequence of the gene from Pseudomonas diminuta MG encoding parathion hydrolase was determined, and a single open reading frame located. Comparison of the deduced N-terminal amino acid sequence with that determined by protein sequence analysis of the enzyme from P. diminuta MG indicated that parathion hydrolase is synthesized as a 365 amino acid precursor from which 29 amino acids are removed. Expression of the processed parathion hydrolase coding sequence in Escherichia coli under the control of lambda PL promoter results in the production of high-level enzyme activity. Furthermore, addition of metal salts to the growth medium enhanced specific activity. The N-terminal amino acid sequence, C-terminal amino acid sequence and the amino acid composition of the purified enzyme were in agreement to those expected from the translated DNA sequence.
TL;DR: It is believed that the cellulose-binding domain (CBDCex) of Cex offers a simple, inexpensive and widely applicable method of enzyme immobilization.
Abstract: We have constructed a fusion between the gene (abg) for a β-glucosidase (Abg) of an Agrobacterium sp. and part of the gene (cex) for an exoglucanase (Cex) of Cellulomonas fimi. The abg-cex fusion encodes a fusion protein (Abg-CBDCex) with the cellulose-binding properties of Cex and β-glucosidase activity of Abg. The fusion protein retained 42 percent of its β-glucosidase activity when bound to cellulose. The binding was stable enough to allow continuous hydrolysis of substrate by the enzyme without detectable leaching of the enzyme from the cellulose. We believe that the cellulose-binding domain (CBDCex) of Cex offers a simple, inexpensive and widely applicable method of enzyme immobilization.
TL;DR: Evidence is presented that the intact fusion product has little or no biological activity but biologically active LIF is released by endogenous proteolytic cleavage, suitable for many in vitro cell culture uses and in particular for ES cell culture.
Abstract: Leukemia inhibitory factor [LIF] is a glycoprotein that is able to induce the differentiation of M1 myeloid leukemic cells and prevent the differentiation of murine embryonic stem [ES] cells. This report presents a simple method for the production and purification of large amounts of recombinant murine and human LIF in Escherichia coli. LIF is expressed initially as a fusion product with glutathione Stransferase, separated by a thrombin cleavage site. The fusion product can be rapidly purified on a glutathione-agarose affinity matrix and fully biologically active LIF released from the matrix by cleavage with thrombin. In this form LIF is suitable for many in vitro cell culture uses and in particular for ES cell culture. Further purification to homogeneity of thrombin-released LIF can be achieved by a single fractionation by reversed-phase high performance liquid chromatography; the purified product contains no detectable bacterial endotoxin. Evidence is presented that the intact fusion product has little or no biological activity but biologically active LIF is released by endogenous proteolytic cleavage.
TL;DR: Protoplasts from adult leaves of lettuce are amenable to direct transformation under the same general conditions as those employed for tobacco protoplasts and provide new opportunities in some specific strategies for lettuce breeding.
Abstract: Transgenic plants of lettuce (Lactuca sativa) obtained through electroporation of protoplasts
TL;DR: Stable clones were obtained that secrete levels of human antibody in either open suspension cultures or within microcapsules that compare favorably with those of murine hybridomas.
Abstract: Mouse-human heterohybridoma cell line, GF4/1.1, secretes a human anti-tetanus toxoid (anti-TT) antibody of the γ3 isotype. This line was used as a source of mRNA for the construction of cDNA libraries and the subsequent isolation of the sequences encoding the human heavy and light chains. Expression vectors containing the cDNAs were constructed and introduced into a non-Ig producing, murine hybridoma cell line, together with a dihy-drofolate reductase (dhfr) selectable marker gene. Transfected cells producing human anti-TT antibody were grown in medium with increasing concentrations of the selecting drug (methotrexate). The level of antibody secretion was tested in cells that had adapted to the higher drug concentration and was found to increase in parallel with increases in the steady-state level of Ig mRNA. This increased productivity was not associated with detectable levels of gene amplification. Stable clones were obtained that secrete levels of human antibody in either open suspension cultures or within microcapsules that compare favorably with those of murine hybridomas.
TL;DR: Les problemes lies a l'evolution possible des plantes manipulees genetiquement vers un phenotype «mauvaise-herbes», sont abordes.
Abstract: Les problemes lies a l'evolution possible des plantes manipulees genetiquement vers un phenotype «mauvaise-herbes», sont abordes. Les caracteristiques genetiques intervenant dans ce processus sont etudiees. Les connaissances obtenues en malherbologie sont utilisees pour essayer de comprendre le processus
TL;DR: Plants of Arachis hypogea cultivar SB-11 are regenerated from immature zygotic embryo axis via direct somatic embryogenesis without an intermediate callus stage and 2,4-dichlorophenoxy acetic acid was essential for this response.
Abstract: We have regenerated plants of Arachis hypogea cultivar SB-11 from immature zygotic embryo axis via direct somatic embryogenesis without an intermediate callus stage. Induction and maturation of the somatic embryos was achieved on the same medium. 2,4-dichlorophenoxy acetic acid (2,4-D) was essential for this response. Both the size of the immature zygotic embryo axis and the concentration of 2,4-D were important for direct somatic embryogenesis. Embryos germinated in hormone free medium, and plants survived in a sand:soil mixture.
TL;DR: The use of several independent strategies to avoid degradation of recombinant proteins in heterologous hosts are demonstrated and high levels of soluble and active fusion protein could be produced.
Abstract: We have used a fusion protein between staphylococcal protein A and E. coli β–galactosidase as a model system to investigate different approaches to stabilize recombinant gene products. First, growth conditions were adapted to preferentially produce insoluble inclusion bodies. This material was resistant to proteolysis, but no active fusion protein could be recovered. Secondly, biochemical characterization revealed that the soluble fusion protein was cleaved by a membrane–bound protease at a Lys–Arg peptide bound in the linker region. A new linker region was therefore engineered lacking the protease sensitive sequence. The new fusion protein was found to be resistant to degradation and high levels of soluble and active fusion protein could be produced. Finally, a mutant E. coli strain lacking the outer membrane protease OmpT was tested as a production strain, and a stable fusion protein could be recovered. These results demonstrate the use of several independent strategies to avoid degradation of recombinant proteins in heterologous hosts.