Plant seed oil-bodies as an immobilization matrix for a recombinant xylanase from the rumen fungus Neocallimastix patriciarum

Canola seed oil-bodies were investigated as a production vehicle and immobilization matrix for xylanases. A recombinant xynC gene from Neocallimastix patriciarum encoding a xylanase (XynC) was fused to an oleosin coding sequence suitable for targeting the xylanase to the oil-body membrane. This fusion gene was introduced into Brassica napus using Agrobacterium-mediated transformation. Transgenic Canola plants were obtained expressing xylanase which was targeted to the oil-bodies of seeds as shown by analysis with XynC-specific antibodies. Oil-bodies extracted from transgenic seeds exhibited xylanase activity, indicating the immobilization of XynC on the surface of oil bodies and the functioning of the xylanase as a fusion protein. The immobilized XynC retained its optimal temperature, K_m value and specificity. However, it exhibited reduced sensitivity to pH. Furthermore, it was shown that the enzyme immobilized on oil-bodies could be recycled by flotation several times without loss of activity.     

Production of biologically active hirudin in plant seeds using oleosin partitioning

A plant oleosin was used as a carrier for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin carrier. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

Phloem transport of amino acids in two Brassica napus L. genotypes and one B. carinata genotype in relation to their seed protein content

 In order to investigate the relationship between the amino acid concentration in the phloem sap of leaves and the protein content in seeds, two Brassica napus genotypes and one B. carinata genotype with low, medium and high seed protein contents were analyzed. Phloem sap was collected from the B. napus winter rapeseed breeding line DSV15 with 19% protein of dry weight in the seeds, the spring cultivar ‘Duplo’ with 25% protein in the seeds and from the B. carinata line BRA1151/90 with 39% protein in the seeds by using the aphid-stylet technique. The total amino acid contents measured in the phloem varied considerably among the three genotypes analysed, and correlated positively with their respective seed protein contents. The total amino acid-to-sucrose ratio was lowest in B. napus line DSV15 which had the lowest seed protein content and highest in the B. carinata line BRA1151/90 which had the highest seed protein content. The amino-N translocation in the phloem during the light period was about 2-fold higher in the B. carinata line BRA1151/90 than in the B. napus lines Dulpo and DSV15. Predominant amino acids in the phloem were glutamine and glutamate, followed by serine, aspartate, and threonine. The amino acid patterns in the leaves resembled those in the phloem, although their absolute concentrations were higher in the phloem than in the cytosol of mesophyll tissue. Furthermore, the concentration gradient of amino acids between the cytosol of mesophyll cells and the phloem was higher in the B. carinata line BRA1151/90 than in the B. napus lines Duplo and DSV15. These results lead to the conclusion that the phloem translocation of amino-N and the phloem loading process of amino acids are decisive factors for the protein content in the seeds of Brassica species. Received: 28 November 1999 / Accepted: 10 April 2000     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana

 Full-length and N-terminal deletions of a sunflower (Helianthus annuus L.) oleosin protein were expressed ectopically in transgenic Arabidopsis thaliana (L.) Heynh. Immunological detection of the sunflower protein revealed that it accumulated in a range of non-oil-storing tissues, including leaves, roots and petals. This accumulation was shown to result from deposition in the microsomal membrane fraction. Expression in oil-storing tissues (such as seeds) of oleosin N-terminal deletions revealed impaired transfer from the endoplasmic reticulum to the oil body. In non-oil-storing tissues, accumulation in the microsomal membrane fraction was progressively reduced by N-terminal deletion. These data confirm the role of the endomembrane system in the targeting of the oleosin and its intimate relationship with oil-body biogenesis. Received: 26 August 1999 / Accepted: 4 October 1999     

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997     

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata

Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%). Received: 9 April 1997 / Revision received: 3 July 1997 / Accepted: 30 July 1997     

Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds

The 3′-UTR of the FAD2 gene from Brassica carinata was cloned by PCR and used to prepare an intron-spliced hairpin RNA (ihpRNA) construct. Compared to that of the wild type (WT) background, this construct, when expressed in B. carinata, resulted in a high degree of FAD2 gene silencing accompanied by strong increases of up to 16 and 10% in oleic acid and erucic acid proportions, respectively. The increase in 18:1 was accompanied by a concomitant proportional reduction in 18:2. A second construct containing ihpRNA targeted to the endogenous FAD2 gene in addition to the heterologous Crambe abyssinica FAE gene under the control of seed specific napin promoter, was used to transform B. carinata. This approach resulted in an even greater increase in erucic acid proportions, by up to 16% in T₁ segregating seeds as compared to that of the WT control. To our knowledge, this is currently the highest accumulation of erucic acid achieved in B. carinata seeds using transgenic approaches, making it an increasingly-attractive alternative to high erucic B. napus cultivars as an industrial oil crop. Database: The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank under accession number DQ250814.     

Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human β-glucuronidase

 We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human β-glucuronidase by recombinant DNA technology. The sequences encoding the murine monoclonal antibody 323/A3 light- and heavy-chain variable genes were joined by a synthetic sequence encoding a 15-amino-acid linker and combined with human β-glucuronidase by a synthetic sequence encoding a 6-amino-acid linker. The construct was placed under the control of the cytomegalovirus promotor and expressed in COS-7 cells. The yield of active fusion protein was 10 ng/ml transfectoma supernatant. Antibody affinity, antibody specificity and enzyme activity were fully retained by the fusion protein. Biochemical characterization of the fusion protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a molecular mass of 100 kDa under denaturing conditions. Gel-filtration analysis indicated that the enzymatically active form is a tetramer of approximately 400 kDa. The non-toxic prodrug N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate was activated to the cytotoxic drug doxorubicin by the fusion protein with a hydrolysis rate similar to that of human β-glucuronidase. The growth inhibition of tumor cells coated with the fusion protein and exposed to prodrug was similar to that obtained with doxorubicin. This study shows the feasibility of constructing eukaryotic fusion proteins consisting of a single-chain antibody and human β-glucuronidase for use in the specific activation of anticancer prodrugs. Received: 5 June 1997 / Accepted: 25 October 1997     

A study on the effects of food quantity and quality on glutathione S-transferase (GST) activity and growth rate parameters of <Emphasis Type="Italic">Daphnia</Emphasis><Emphasis Type="Italic">carinata</Emphasis> varying in age

The adverse influences of insufficient food and toxins on Daphnia carinata’s body growth, reproduction and tolerance were investigated in the laboratory. Different concentrations of Scendesmus obliquus and a mixture of S. obliquus and microcystin (MC)-containing Microcystis aeruginosa PCC7820 were used to feed D. carinata. Glutathione S-transferase (GST) activity towards five chemical compounds (substrates) was measured and used as an indicator of their tolerance. Body growth rate and clutch size of D. carinata decreased with declined concentration or decreased proportion of S. obliquus in the diet. GST activity decreased with ageing in D. carinata. However, GST activity to several chemical compounds increased when food quantity or food quality decreased. Adult D. carinata had a lower GST activity towards p-nitrophenenzyl chloride (PNBC) than juveniles and exhibited a sharp decline (P < 0.001) in GST activity towards PNBC as the animals aged. It is suggested that the age-specific decrease in GST activity is a possible mechanism for the high morality of adult D. carinata in the summer of eutrophic lakes.     

Expression of biologically active human insulin-like growth factor 1 in Arabidopsis thaliana seeds via oleosin fusion technology

Novel protein expression in plant-based systems has become an important tool in producing and studying therapeutic proteins. Among many plant-based systems developed so far, oleosin fusion technology is one of the most cost-effective and convenient methods. In this study, an important therapeutic protein, human insulin-like growth factor 1 (hIGF-1), was expressed in Arabidopsis thaliana seeds via this technology. The plant bias codon usage-optimized hIGF-1 gene was fused to the C-terminal of A. thaliana 18.5 kDa oleosin gene, and the fusion gene driven by an oleosin promoter was transferred into A. thaliana ecotype Col-0. The accumulation of oleosin-hIGF-1 fusion protein in transgenic seeds was up to 0.75% of total seed protein (TSP) and the expression level of hIGF-1 was 0.17% of the TSP, which was eight times higher than previously reported using other plant-based hIGF-1 production systems. The biological activity of the hIGF-1 as an oleosin-hIGF-1 fusion protein in vitro was demonstrated by using human SH-SY5Y neuroblastoma cells.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l⁻¹ and 7 g fusion glucagon l⁻¹. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content. Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997     

Tunable nano‐oleosomes derived from engineered Yarrowia lipolytica

Oleosomes are discrete organelles filled with neutral lipids surrounded by a protein‐embedded phospholipid monolayer. Their simple yet robust structure, as well as their amenability to biological, chemical, and physical processing, can be exploited for various biotechnology applications. In this study, we report facile biosynthesis of functionalized oleosomes within oleaginous yeast Yarrowia lipolytica, through expression of oleosin fusion proteins. By fusing a cDNA clone of a sesame oleosin with either the coding sequence of a red fluorescent protein mCherry or a cellulosomal scaffolding protein cohesin from Clostridium cellulolyticum, these oleosin‐fusion proteins were efficiently expressed and specifically targeted to and anchored on the surface of the oleosomes within the Y. lipolytica cells. The engineered oleosomes can be easily separated from the Y. lipolytica cell extract via floating centrifugation and both mCherry and cohesin domains are shown to be functional. Upon sonication, the engineered Yarrowia oleosomes exhibit a mean diameter of 200–300 nm and are found to be highly stable. The feasibility of co‐displaying multiple proteins on the Yarrowia oleosomes was demonstrated by incubating cohesin‐displaying oleosomes with different dockerin‐fusion proteins. Based on this strategy, engineered oleosomes with both cell‐targeting and reporting activities were created and shown to be functional. Taken together, the Yarrowia oleosome surface display system in which oleosin serves as an efficient membrane anchor motif shows great promise as a simple platform for creating tunable nanoparticles. Biotechnol. Bioeng. 2013; 110: 702–710. © 2012 Wiley Periodicals, Inc.     

Production and cytogenetics of the intergeneric hybrids Brassica juncea×Orychophragmus violaceus and B. carinata×O. violaceus

 Intergeneric hybrids between Brassica juncea (2n=36), B. carinata (2n=34) and Orychophragmus violaceus (2n=24) were produced when B. juncea and B. carinata cultivars were used as female parents. The hybrids between B. juncea and O. violaceus had an intermediate morphology except for petal colour and were partially fertile. The hybrids between B. carinata and O. violaceus had a matroclinous morphology and were nearly fertile. Cytological analysis of the hybrids and their progenies gave the following results. (1) In the hybrids between B. juncea and O. violaceus, the somatic tissues of the roots, leaves and styles were mixoploid (2n=12–42), and cells with 24, 30 or 36 chromosomes were the most frequent. Based on the recorded numbers and behaviour of the mitotic and meiotic chromosomes, complete and partial separation of the parental genomes was proposed to have occurred during mitosis. This resulted in the occurrence of cells with possibly complete and incomplete complements of the parental species and cells with parental complements and some additional chromosomes from the other parent. (2)  Pollen mother cells (PMCs) possibly with both parental chromosome complements, only B. juncea chromosomes or a complete B. juncea complement with additional O. violaceus chromosomes were more competitive in entering meiosis. The majority of fertile gametes were deduced to have been produced by PMCs with a B. juncea complement with or without additional O. violaceus chromosomes. (3) The progeny plants from selfed hybrids between B. juncea and O. violaceus were morphologically either of a B. juncea, hybrid or variable type. Cytologically they were grouped into six types according to the frequencies of cells with various chromosome numbers. All of the plants except 2 which constituted two types, were mixoploids, composed of cells with various chromosome numbers, mainly in a certain serial range. (4) The hybrid plants between B. carinata and O. violaceus were mixoploids with chromosome numbers in the range of 12–34, and cells with 2n=34 were the most frequent. The main categories of PMCs with 17 bivalents at metaphase I and 17 : 17 segregations at anaphase I contributed to the high fertility of the hybrids and the fact that their progeny after selfing were mainly plants with 2n=34. Somatic and meiotic separation of the parental genomes was proposed to have occurred in the hybrids between B. carinata and O. violaceus. (5) Mitotic and meiotic elimination of what could be O. violaceus chromosomes might also have contributed to the observed mitotic and meiotic cell types in the two kinds of hybrids studied. Finally, the possible mechanisms behind these cytological observations and their potential in the production of Brassica aneuploids were discussed. Received: 4 February 1997/Accepted: 29 July 1997     

Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.)

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria. Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997