Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Species-specific size expansion and molecular evolution of the oleosins in angiosperms

Oleosins are hydrophobic plant proteins thought to be important for the formation of oil bodies, which supply energy for seed germination and subsequent seedling growth. To better understand the evolutionary history and diversity of the oleosin gene family in plants, especially angiosperms, we systematically investigated the molecular evolution of this family using eight representative angiosperm species. A total of 73 oleosin members were identified, with six members in each of four monocot species and a greater but variable number in the four eudicots. A phylogenetic analysis revealed that the angiosperm oleosin genes belonged to three monophyletic lineages. Species-specific gene duplications, caused mainly by segmental duplication, led to the great expansion of oleosin genes and occurred frequently in eudicots after the monocot–eudicot divergence. Functional divergence analyses indicate that significant amino acid site-specific selective constraints acted on the different clades of oleosins. Adaptive evolution analyses demonstrate that oleosin genes were subject to strong purifying selection after their species-specific duplications and that rapid evolution occurred with a high degree of evolutionary dynamics in the pollen-specific oleosin genes. In conclusion, this study serves as a foundation for genome-wide analyses of the oleosins. These findings provide insight into the function and evolution of this gene family in angiosperms and pave the way for studies in other plants.     

Brassica napus pollen oleosins possess a characteristic C-terminal domain

The sequences of Brassica napus L. pollen oleosins have been determined and examined. Contrary to a recent report, inferred primary sequences of pollen oleosins do include a unique C-terminal domain characterised by the presence of a repetitive motif of three alanine and one proline residue (AAAP). This motif appears to be present in all oleosins expressed in pollen, but not in oleosins from other tissues.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Genes encoding oleosins in maize kernel of inbreds Mo17 and B73

We have investigated all three oleosin genes which are expressed in the kernel of maize (Zea mays L., Mo17). Oleosin genes, ole16, ole17, and ole18, encode OLE16, OLE17, and OLE18, respectively, in proportional amounts of approximately 2:1:1 in isolated oil bodies. None of the three genes has an intron or a sequence encoding an N-terminal signal peptide. The three genes are expressed coordinately during seed maturation, and their encoded oleosins are present in similar proportional amounts in oil bodies isolated from the embryonic axis, scutellum, and aleurone layer. OLE16 represents one oleosin isoform, whereas OLE17 and OLE18 are close members of another oleosin isoform. ole16 and ole18 have been mapped to single loci on chromosomes 2 (near b1 gene) and 5S (near phya2), respectively. We predict that ole17 is located on chromosome 1 (near phya1), in a chromosomal segment duplicated on chromosome 5.     

The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis

We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.     

A lipid droplet protein of Nannochloropsis with functions partially analogous to plant oleosins

As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.     

盐生植物及其开发利用

当前,研究盐生植物的抗盐机理,开发抗盐碱种质资源是一个全球性热点。对盐胁迫对植物的危害、盐生植物的种类及盐生植物的开发利用等方面进行概述,以期为盐生植物的开发利用和进一步研究提供参考。     

香椿资源的研究、开发现状

香椿是我国特有的树种,具有很高的经济价值。对香椿的营养成分、活性成分、临床应用等研究进行综述,并对进一步深入研究开发提出了合理建议。     

Sensory exploitation and sexual conflict

Much of the literature on male-female coevolution concerns the processes by which male traits and female preferences for these can coevolve and be maintained by selection. There has been less explicit focus on the origin of male traits and female preferences. Here, I argue that it is important to distinguish origin from subsequent coevolution and that insights into the origin can help us appreciate the relative roles of various coevolutionary processes for the evolution of diversity in sexual dimorphism. I delineate four distinct scenarios for the origin of male traits and female preferences that build on past contributions, two of which are based on pre-existing variation in quality indicators among males and two on exploitation of pre-existing sensory biases among females. Recent empirical research, and theoretical models, suggest that origin by sensory exploitation has been widespread. I argue that this points to a key, but perhaps transient, role for sexually antagonistic coevolution (SAC) in the subsequent evolutionary elaboration of sexual traits, because (i) sensory exploitation is often likely to be initially costly for individuals of the exploited sex and (ii) the subsequent evolution of resistance to sensory exploitation should often be associated with costs due to selective constraints. A review of a few case studies is used to illustrate these points. Empirical data directly relevant to the costs of being sensory exploited and the costs of evolving resistance is largely lacking, and I stress that such data would help determining the general importance of sexual conflict and SAC for the evolution of sexual dimorphism.     

The exploitation of mutualisms

Mutualisms (interspecific cooperative interactions) are ubiquitously exploited by organisms that obtain the benefits mutualists offer, while delivering no benefits in return. The natural history of these exploiters is well-described, but relatively little effort has yet been devoted to analysing their ecological or evolutionary significance for mutualism. Exploitation is not a unitary phenomenon, but a set of loosely related phenomena: exploiters may follow mixed strategies or pure strategies at either the species or individual level, may or may not be derived from mutualists, and may or may not inflict significant costs on mutualisms. The evolutionary implications of these different forms of exploitation, especially the threats they pose to the stability of mutualism, have as yet been minimally explored. Studies of this issue are usually framed in terms of a "temptation to defect" that generates a destabilizing conflict of interest between partners. I argue that this idea is in fact rather inappropriate for interpreting most observed forms of exploitation in mutualisms. I suggest several alternative and testable ideas for how mutualism can persist in the face of exploitation.     

Development and exploitation of CK2 inhibitors

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (K_i = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (K_i = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (K_i = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (K_i = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs.     

Biotechnological exploitation of bacteriophage research

The experimentally amenable nature of phage and their use in testing fundamental biological questions have meant that phage research has had a profound effect on modern molecular biology. Phage research has also fuelled multiple biotechnological developments. For example, phage display has recently been harnessed in a multidisciplinary approach for the generation of novel nanotechnologies. In addition, with the emerging threat of antibiotic-resistant bacterial infections, phage have begun to provide technologies to combat these problems. Finally, recent data acquired from genome sequencing and advances in phage biology research have aided the development of phage-derived bacterial detection and treatment strategies in addition to methods to control the detrimental effects of phage in industry. Here, we examine the promising uses of phage in these important areas of biotechnology.