基因工程培育可恢复的植物雄性不育系的研究进展

植物雄性不育是植物杂种优势利用的资源, 具有重要的生产利用价值。植物雄性不育可从自然突变、人工诱变和远缘杂交中发现, 现在可通过细胞工程和基因工程等方法来创造。文章综述了利用基因工程方法制备雄性不育品系及其相应的育性恢复策略, 分为“单组分策略”和“双组分策略”。其中利用“单组分策略”制备的不育植株是条件型雄性不育(可逆转的雄性不育), 它能在特定的条件下实现雄性可育与不育的转换, 实践中可直接作为两用系(不育系和保持系)用于两系法杂交制种; “双组分策略”是利用基因互作和亲本杂交直接培育雄性不育系, 或利用基因互作原理分别研制不育系和恢复系, 用于三系法生产杂交种。文章分析了 “单组分策略”和“双组分策略”的基因工程方法培育雄性不育系及其相应育性恢复策略优缺点, 对以上两种技术路线在实际应用中的现状作了分析和展望。     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Lattice Structure of Cytoplasmic Microtubules in a Cultured Mammalian Cell

Tubulin can polymerize in two distinct arrangements: “B-lattices,” in which the α-tubulins of one protofilament lie next to α-tubulins in the neighboring protofilaments, or the “A” configuration, where α-tubulins lie beside β-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this “tip-associating protein,” may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a “seam,” one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins.     

Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site

Extracellular secretion of recombinant proteins from plant cell suspension culture will simplify the protein purification procedure and greatly reduce the production cost. Our early work indicated that presence of hydroxyproline-O-glycosylation at the C- or N-terminus of the target protein boosted the secreted yields in the culture medium. Inspired by early successes, we tested the possibility of introducing an N-glycosylation site to facilitate the secretion of human growth hormone (hGH) from cultured tobacco cells. Three N-glycosylated hGH fusion proteins, designated NAS-EK-hGH, NAS-Kex2-hGH and hGH-NAS, were expressed in tobacco BY-2 cells. Where NAS denotes the “Asn-Ala-Ser” consensus sequence for N-glycosylation; EK denotes an enterokinase cleavage site and Kex2 a sequence to be cleaved by a Golgi-localized Kex2p-like protease. Our results indicated that a single N-glycan attached either at the N-terminus or C-terminus of hGH correlated with enhanced extracellular accumulation of the transgenic proteins; the secreted yield of NAS-EK-hGH and hGH-NAS was 70-90 fold greater than the control targeted, non-glycosylated hGH. NAS-Kex2-hGH was subject to partial cleavage of the N-glycan tag at the Kex2 site in Golgi apparatus, and therefore gave lower yields than the other two constructs.     

A polypeptide "building block" for the β-trefoil fold identified by "top-down symmetric deconstruction"

Fibroblast growth factor-1, a member of the 3-fold symmetric β-trefoil fold, was subjected to a series of symmetric constraint mutations in a process termed “top-down symmetric deconstruction.” The mutations enforced a cumulative exact 3-fold symmetry upon symmetrically equivalent positions within the protein and were combined with a stability screen. This process culminated in a β-trefoil protein with exact 3-fold primary-structure symmetry that exhibited excellent folding and stability properties. Subsequent fragmentation of the repeating primary-structure motif yielded a 42-residue polypeptide capable of spontaneous assembly as a homotrimer, producing a thermostable β-trefoil architecture. The results show that despite pronounced reduction in sequence complexity, pure symmetry in the design of a foldable, thermostable β-trefoil fold is possible. The top-down symmetric deconstruction approach provides a novel alternative means to successfully identify a useful polypeptide “building block” for subsequent “bottom-up” de novo design of target protein architecture.     

A streamlined protocol for emulsion polymerase chain reaction and subsequent purification

We report the development of a novel ELISA platform to quantitate hepatitis B virus X (HBx) protein refolding yields, which is critical for rational design and scaleup of aHBx bioprocess. HBx refolding yields were measured by determining the amount of HBx bound to immobilized GST–p53 on a “reduced glutathione”-functionalized maleimide surface. Refolding yields were distinguished from soluble yields, which were determined by measuring total HBx protein bound to a maleimide surface under reducing conditions. This platform is amenable to scaleup, and will expedite HBx production for structural and clinical studies, leading to the development of HBx-based therapy for liver cancer.     

New design platform for malonyl-CoA-acyl carrier protein transacylase

Malonyl-CoA-acyl carrier protein transacylase (MCAT) transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), and since malonyl-ACP is a key building block for fatty-acid biosynthesis it is considered as a promising antibacterial target. The crystal structures of MCAT from Staphylococcus aureus and Streptococcus pneumoniae have been determined at 1.46 and 2.1 Å resolution, respectively. In the SaMCAT structure, the N-terminal expression peptide of a neighboring molecule running in the opposite direction of malonyl-CoA makes extensive interactions with the highly conserved “Gly-Gln-Gly-Ser-Gln” stretch, suggesting a new design platform. Mutagenesis results suggest that Ser91 and His199 are the catalytic dyad.     

Changes in protein synthesis during the initial stage of life of tobacco protoplasts

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts (in vitro labeling) and into leaves, from which the protoplasts were isolated later (in situ labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.     

Foaming of proteins: New prospects for enzyme purification processes

Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the “White Biotechnologies” for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique.     

Plant‐derived recombinant human serum transferrin demonstrates multiple functions

Human serum transferrin (hTf) is the major iron‐binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high‐quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 μg/g fresh leaf weight). Furthermore, plant‐derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum‐free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell‐specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes. To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon‐like peptide 1 (GLP‐1) or its derivative in plants. Here, we show that plant‐derived hTf‐GLP‐1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro.     

High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures

The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.     

Intercellular exchange of proteins: The immune cell habit of sharing

The recent recognition of new types of cell-cell communication pathways challenges classic theories of cell autonomy. Evidence of functional “proteome mixing” among interacting cells, particularly immune cells, supports the notion that no cell is an island, and that even these “unsplittable” units are actually non-autonomous. We summarize various mechanisms of intercellular transfer of proteins—trans-endocytosis, trogocytosis, exosomal transport, shuttle through nanotubes, and cell-contact-dependent intercellular transfer of intracellular proteins including oncogenic Ras. These phenomena suggest exciting new possibilities for proteome research, focusing on system-level proteomics that characterize cell contents and functions in the context of intercellular protein transfer.     

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.     

Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells

A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as “non-dendritic cells” or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these “junk cells” are actually qualified dendritic cells.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

The sense of water in the blowfly Protophormia terraenovae

The gustatory system of the blowfly, Protophormia terraenovae, is a relatively simple biological model for studies on chemosensory input and behavioral output. It appears to have renewed interest as a model for studies on the role of water channels, namely aquaporins or aquaglyceroporins, in water detection. To this end, we investigated the presence of water channels, their role in “water” and “salt” cell responsiveness and the transduction mechanism involved. For the first time our electrophysiological results point to the presence of an aquaglyceroporin in the chemoreceptor membrane of the “water” cell in the blowfly taste chemosensilla whose transduction mechanism ultimately involves an intracellular calcium increase and consequently cell depolarization. This hypothesis is also supported by calcium imaging data following proper stimulation. This mechanism is triggered by “water” cell stimulation with hypotonic solutions and/or solutes such as glycerol which crosses the membrane by way of aquaglyceroporins. Behavioral output indicates that the “sense” of water in blowflies is definitely not dependent on the “water” cell only, but also on the “salt” cell sensitivity. These findings also hypothesize a new role for aquaglyceroporin in spiking cell excitability.     

兼职功能蛋白

兼职功能蛋白(moonlighting proteins)是指一类具有两种或两种以上功能的蛋白,且这些功能间没有直接相关性,此类蛋白能够通过多种形式转换其功能.随着科学研究的深入,越来越多的已知功能的蛋白被发现具有新型兼职功能,其兼职功能对生物体的意义绝不亚于其所谓本职功能.兼职功能蛋白的发现大大拓展了基因、蛋白质与生理功能一一对应的传统观念;特别是最近研究发现一类叫做无固有结构蛋白(intrinsically unstructured proteins,IUPs)表现出多功能性,向"蛋白质的功能等视于确定的三维结构"的经典定律发起了挑战.本文综述了兼职功能蛋白的功能转换机制、进化历程、研究方法等方面的最新研究进展,同时对兼职功能蛋白给生命科学研究带来的新思路和新挑战进行了深入的讨论.     

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

Low‐yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline‐O‐glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY‐2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of ”Ser‐Pro“ dipeptide, or (SP)₃₂, to study cell growth and protein secretion, culture scale‐up, and establishment of perfusion cultures for continuous production. The BY‐2 cells accumulated low levels of cell biomass (～7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)₃₂‐tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY‐2 cells cultured in a 5‐L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day^?1, generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures.