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.     

Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion

A variety of single-chain variable fragments (scFv) that had been previously developed to the surface epitopes of infective Trichostrongylus colubriformis L3 pathogenic gut nematodes of sheep were fused to a trimeric version of polyoleosin (three head-to-tail repeats of oleosin) and expressed in planta under the control of an Arabidopsis oleosin promoter. The fusion products were found to accumulate in oil bodies (OBs) at the range of 0.25-0.9% of the total seed protein which is comparable with the main 18kDa isoform of Arabidopsis seed oleosin. Immunofluorescence microscopy and immuno-binding were used to demonstrate that it is possible to both purify the recombinant protein via enrichment for OBs as well as use the OBs emulsion to deliver functional recombinant scFv. This work presents a novel fusion strategy platform to boost the productivity and simplify the delivery of recombinant single chain antibodies and other like proteins.     

Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.     

Structural requirements of oleosin domains for subcellular targeting to the oil body.

We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins. We have attempted to study this targeting in vivo using "tagged" oleosins in transgenic plants. Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins. Each was fused in frame to the Escherichia coli uid A gene encoding beta-glucuronidase (GUS). These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation. GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds. It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds. Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting. In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein. Northern blotting revealed that this reduction is not due to differences in mRNA accumulation. Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability.     

Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil-body system

Royalisin found in the royal jelly of Apis mellifera is an antimicrobial peptide (AMP). It has a molecular weight of 5.5 kDa, which contains six cysteine residues. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as two oleosin-fusion proteins for preparation of its antibodies and functional purification. The recombinant royalisin, fused with oleosin central hydrophobic domain in both N- and C-termini, was reconstituted with triacylglycerol and phospholipids to form artificial oil bodies (AOBs). The AOBs were then purified to raise the antibodies. These antibodies could recognize both the native and recombinant royalisins, but not oleosin. Another oleosin-intein S-fusion protein was purified by AOBs system, and royalisin was subsequently released from the AOBs through self-splicing of the intein. The recombinant royalisin exhibited high antibacterial activity, which suggested that it was refolded to its functional structure. These results demonstrated that AOBs system is an efficient method to functionally express and purify small AMPs. In addition, it also provides a facile platform for the production of antibodies against small peptides.     

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization

Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.     

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.     

Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.     

异源过表达水稻OsSAPP3基因促进拟南芥叶片衰老

蛋白磷酸酶催化的蛋白质可逆磷酸化反应是叶片衰老的关键环节。该研究筛选并克隆了1个新的参与水稻(Oryza sativa)叶片衰老调控的PP2C基因OsSAPP3。研究表明, OsSAPP3的启动子在Pro_OsSAPP3-GUS转基因拟南芥(Arabidopsis thaliana)的莲座叶中有活性, 并且活性以依赖叶龄方式增加。利用CaMV 35S启动子驱动组成型异源过表达OsSAPP3导致转基因拟南芥无法正常生长。用可诱导型启动子GVG系统驱动OsSAPP3异源过表达导致转基因拟南芥出现莲座叶变小、数量增加、叶片早衰及抽薹开花提前等早衰表型。外源诱导OsSAPP3基因异源过表达后, 利用实时荧光定量PCR检测到SAG12、WRKY6和NAC2等衰老标志基因显著上调表达。研究结果表明, OsSAPP3是参与水稻叶片衰老的正向调控因子。     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

Expression of Human FGF18 by Fusion with Oleosin in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds

Expression of recombinant human fibroblast growth factor 18 (hFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, but they are difficult, expensive. The expression of recombinant proteins fused to oleosin protein have distinct advantages, such as safety, ease, low cost. So we have expressed hFGF18 fused to oleosin protein in the oil bodies of Arabidopsis thaliana (A. thaliana) and screen the proliferation effect of NIH3T3 cells. The vector of oleosinhFGF18 fusion gene was constructed and transformed into wild A. thaliana. Transformed A. thaliana lines were obtained by the floral dip method and confirmed using polymerase chain reaction (PCR). The PCR results indicated that the oleosin-hFGF18 fusion gene was integrated into the A. thaliana genome. The oil bodies expression of oleosin-hFGF18 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biological activity showed that oil bodies expressing oleosin-hFGF18 could stimulate the proliferation of NIH3T3 cells.