Expression of functionally active sialylated human erythropoietin in plants

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92.5 ±3.0) g/L and 0.29±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92. 5±3.0) g/L and 0.29 ±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

In vivo glyco-engineered antibody with improved lytic potential produced by an innovative non-mammalian expression system

Recent studies have demonstrated that the reduction of the core fucosylation on N-glycans of human IgGs is responsible for a clearly enhanced antibody-dependent cellular cytotoxicity (ADCC). This finding might give access to improved active therapeutic antibodies. Here, the expression of the tumor antigen-specific antibody IGN311 was performed in a glyco-optimized strain of the moss Physcomitrella patens. Removal of plant specific N-glycan structures in this plant expression host was achieved by targeted knockout of corresponding genes and included quantitative elimination of core fucosylation. Antibodies transiently expressed and secreted by such genetically modified moss protoplasts assembled correctly, showed an unaltered antigen-binding affinity and, in extensive tests, revealed an up to 40-fold enhanced ADCC. Thus, the glyco-engineered moss-based transient expression platform combines a rapid technology with the subsequent analysis of glycooptimized therapeutics with regard to advanced properties.     

A novel aspartic proteinase is targeted to the secretory pathway and to the vacuole in the moss Physcomitrella patens

In seed plants aspartic proteases (APs) are known to reside in storage vacuoles. Targeting to this compartment is provoked by a secretory signal peptide and the plant-specific insert (PSI). In order to study secretory and vacuolar targeting in a seedless plant, the moss Physcomitrella patens, we isolated a cDNA encoding PpAP1, a novel aspartic proteinase. Sequence alignment with other members of the family of plant APs (EC 3.4.23) revealed a high overall identity and the Pfam motifs for aspartic proteinase and PSI were clearly recognised. In phylogenetic analysis PpAP1 was placed at a very basal position outside of the bigger clusters. Protoplasts transiently expressing the PpAP1 signal peptide fused to GFP showed fluorescence in a well-developed ER-Golgi network. A C-terminal fusion of GFP to the entire PpAP1 protein showed vacuolar fluorescence in transiently transfected protoplasts. Therefore, the vacuole is apparently the in-vivo target for PpAP1. In this study the three-dimensional peculiarity of the endomembrane continuum of ER and Golgi was visualised in a seedless plant for the first time. Above all the functionality of the secretory and the vacuolar targeting signals make them become useful tools for biotechnological approaches.     

Involvement of an acid phosphatase on cell wall regeneration of tobacco protoplasts

Tobacco protoplasts begin to regenerate their own cell walls, the major components of which are β-glucans, soon after they are transferred into an adequate medium. During the cell wall regeneration the protoplasts secrete two isoforms of acid phosphatase (APase) in time-dependent manner. We determined that one of the isoforms, the Brefeldin A (BFA) sensitive one, is the cell wall resident APase (WP-II) by immunoblotting of the isoform with anti-WP-II antibody. We hypothesized that the WP-II may participate in the deposition of β-glucan microfibrils on the protoplast surface during cell wall regeneration. In order to examine this hypothesis, the protoplasts were cultivated in the cell wall regeneration medium containing the same amount of the BFA-sensitive APase (230 µg protein) as is secreted by the observed number of protoplasts (1.4 × 10⁵ protoplasts) per plate (30-mm-diameter) during a 3-h cultivation after transfer to the cell wall regeneration medium. The addition of WP-II to the cell wall regeneration medium stimulated the deposition of β-glucan microfibrils on the surface of the protoplasts during cell wall regeneration. To determine the stimulative effect of the 60 kDa polypeptide of WP-II, protoplasts were cultivated in the medium containing the amount of anti-WP-II IgG (230 µg protein) equivalent to the BFA-sensitive APase. These results suggested that the 60 kDa polypeptide of WP-II is the BFA-sensitive APase which is responsible for the enhanced deposition of β-glucan microfibrils on the surface of the protoplasts.     

Glyco-engineering of moss lacking plant-specific sugar residues

The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific alpha(1,3)-fucose and beta(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal beta(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the alpha(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human beta(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked alpha(1,3)-fucose and beta(1,2)-xylose residues, whereas beta(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretory pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.     

A fast and flexible PEG-mediated transient expression system in plants for high level expression of secreted recombinant proteins

Plant expression systems offer a valuable alternative to traditional systems for the production of recombinant biopharmaceuticals. A highly efficient polyethyleneglycol (PEG)-mediated transient expression system for secreted recombinant proteins in plants has been developed. The human vascular endothelial growth factor 121 (rhVEGF) has been successfully expressed and efficiently secreted into the culture medium by transiently transformed moss protoplasts. In order to obtain secretion efficiency data, different expressed signal peptides were analysed and time course studies were performed with expression constructs containing different promoters. The transformation procedure was optimised for high level expression (up to 10 microg/ml) and successfully performed even with a transgenic glyco-engineered strain lacking plant-specific immunogenic sugar residues in N-glycans. The amount of rhVEGF was produced in such quantity that it allowed for the analysis of biological activity, silver-staining and Western blotting, revealing the correct formation and processing of the homodimer. This fast and flexible transient expression system enables feasibility studies and construct optimisation to be concluded within a few days, thus avoiding the time consuming step of having to generate stably transformed lines.     

人红细胞生成素单克隆抗体的制备、鉴定及应用研究

用rhEPo作为抗原,免疫BALB／c小鼠,取其脾细胞与x63Ag8．653小鼠骨髓瘤细胞融合,再碱性PAGE方法进一步分离并纯化的rhEpo,包被Pvc板,对杂交瘤用ELlSA方法进行筛选,获得两株稳定分泌抗hEPO单抗的杂交瘤细胞株。经鉴定分别属于IgG1、IgG2b,轻链均为k链,Kd分别为5．53×10^-10mol／L和1．34×1O^-10mol／L．用western blot方法证明两者对hEPO具有高度韵专一性．能特异地识别rhEPO和尿源hEPO。所制备单抗可作为亲和层析的配体,用于再生障碍性贫血病人尿中EPO及哺乳类工程细胞所表达的hEPO的分离、纯化,并可用于hEPO的定量检测．     

Recombinant human erythropoietin produced in milk of transgenic pigs

We have developed a line of transgenic swine harboring recombinant human erythropoietin through microinjection into fertilized one cell pig zygotes. Milk from generations F1 and F2 transgenic females was analyzed, and hEPO was detected in milk from all lactating females at concentrations of approximately 877.9+/-92.8 IU/1 ml. The amino acid sequence of rhEPO protein in the transgenic pig milk matched that of commercial rhEPO produced from cultured animal cells. In addition, an F-36 cell line, which proliferates in the presence of hEPO or commercial EPO, was induced to synthesize erythroid by extracts from tg sow milk. This study provides evidence that production of purified rhEPO from transgenic pig milk is a potentially valuable technology, and can be used as a cost-effective alternative in clinical applications as well as providing other clinical advantages.     

Generation of transgenic silkworms for production of erythropoietin in Bombyx mori

There have been many attempts to generate various essential proteins using transformed E. coli systems. However, prokaryote systems are not equipped with the protein maturation mechanisms necessary to generate eukaryotic proteins. In this sense, among the eukaryotes, silkworms have major merits in overcoming the difficulties. Such protein maturation mechanisms are available in silkworms. In this study, a transgenic silkworm producing rhEPO in the cocoon was generated and purified. Specifically, we constructed a transgenic silkworm using a vector system that could be controlled to the next generation. To accomplish this, we microinjected the system into eggs laid during the preblastoderm stage. The rhEPO was then purified from transgenic silkworm cocoons using a Con A affinity column. The biological activity of rhEPO isolated from the cocoon of transgenic silkworms was then assessed in a cell culture system using an EPO-dependent cell line, F-36E. Next, PCR analysis was used to demonstrate that stable gene expression can occur in the embryos of the silkworm, Bombyx. mori. Transgenic silkworms were then selected and observed to ensure that the transgenic silkworm was maintained and transmitted to their progeny. The rhEPO was subsequently purified from the transgenic silkworm cocoon and the electrophoretic pattern of the purified rhEPO revealed a protein band with a molecular weight of approximately 20 kDa. A total of 3 mg of rhEPO was eluted from 10 g of cocoons. The proliferation of F36E cells for 25 ng/ml rhEPO was 1.32, while the proliferation for 2.5 IU/ml hEPO was 1.32. The proliferation of these cells could be induced by commercial hEPO, as well as by rhEPO from transgenic silkworm cocoons. An in vivo test of mice treated with rhEPO revealed relatively high RBC values when compared to normal mice. These results indicated that purified glycosylated EPO from transgenic silkworms had biological activities. Overall, the transgenic silkworm technique will be very useful for the large scale production of proteins for diagnostic and therapeutic purposes.     

人促红细胞生成素基因组基因和cDNA的克隆及其在COS－7细胞中的表达

利用ＰＣＲ技术,从正常人胎肝染色体ＤＮＡ中克隆到长度为１５７２ｂｐ的人促红细胞生成素（ＥＰＯ）基因组基因片段,它包含除第一个外显子和第一个内含子外所有外显子及内含子。再人工合成１３ｂｐ外显子１的编码区,并与１５７２ｂｐ片段拼接,从而得到除第一个内含子的人促红细胞生成素基因组基因。将克隆得到的ＥＰＯ基因插入载体ｐＳＶ２－ｄｈｆｒ得到ｐＳＶ２－ＥＰＯ表达载体,转染ＣＯＳ－７细胞后获得高效表达。利用自行研制的小鼠抗人ＥＰＯ单抗及兔抗人ＥＰＯ多抗,对表达产物进行ＥＬＩＳＡ定量测定,细胞分泌ＥＰＯ量高达２５１±７Ｕ／ｍｌ．Ｋｒｙｓｔａｌ法测得体外生物活性２４１．５±６．５Ｕ／ｍｌ．用ＥＰＯ单抗免疫沉淀结合ＳＤＳ－ＰＡＧＥ对转染细胞的表达产物做了进一步鉴定,清晰地看到了ＥＰＯ条带。从高效表达ＥＰＯ的转染细胞中分离纯化ｍＲＮＡ,用ＲＴ－ＰＣＲ方法扩增并克隆到ＥＰＯ的ｃＤＮＡ,这为ＥＰＯ在其它系统中的表达及ＥＰＯ的功能与结构的研究打下了基础。     

Expression of rat beta(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation

Plant N -linked glycans differ substantially from their mammalian counterparts, mainly with respect to modifications of the core glycan, which typically contains a β(1,2)-xylose and an α(1,3)-fucose. The addition of a bisecting N -acetylglucosamine residue by β(1,4)- N -acetylglucosaminyltransferase III (GnTIII) is known to control the processing of N -linked glycans in mammals, for example by preventing α(1,6)-fucosylation of the core glycan. In order to outcompete plant-specific β(1,2)-xylose and α(1,3)-fucose modifications, rat GnTIII was expressed either with its native localization domain (GnTIII) or with the cytoplasmic tail, transmembrane domain and stem region (CTS) of Arabidopsis thaliana mannosidase II (ManII) (GnTIII^A.th.). Both CTSs targeted enhanced yellow fluorescent protein (eYFP) to a brefeldin A-sensitive compartment, indicative of Golgi localization. GnTIII expression increased the fraction of N -glycans devoid of xylose and fucose from 13% ± 7% in wild-type plants to 60% ± 8% in plants expressing GnTIII^A.th.. N -Glycans of plants expressing rat GnTIII contained three major glycan structures of complex bisected, complex, or hybrid bisected type, accounting for 70%–85% of the total N -glycans. On expression of GnTIII^A.th., N -glycans displayed a higher heterogeneity and were of hybrid type. Co-expression of A. thaliana ManII significantly increased the amount of complex bisected structures relative to the plants expressing GnTIII or GnTIII^A.th., whereas co-expression of human ManII did not redirect the pool of hybrid structures towards complex-type structures. The method described offers the advantage that it can be implemented in any desired plant system for effective removal of β(1,2)-xylose and α(1,3)-fucose from the N -glycan.     

稳定表达促红细胞生或素的重组腺病毒的构建及其对大鼠的促红细胞生成作用

用EPO基因组基因构建了腺病毒质粒型载体psp1B/hEPO,该质粒含有以RSV-LTR为启动子的完整的EPO基因表达盒.单独转染CHO细胞,经暂态表达检测到EPO的表达。用psp1B/hEPO与腺病毒拯救型载体pBHG11共转染293细胞,获得了表达EPO的重组腺病毒AdhEPO.经Southern杂交证实AdhEPO中有EPO表达盒,ELISA检测到了EPO阳性表达.用5×108pfu的AdhEPO给大鼠作一次性肌肉注射,观察到了其促进大鼠红细胞生成的短期效应。在注射后第1,3,5,7,10d分别检测了大鼠的红细胞压积、血红蛋白含量和红细胞计数等指标,发现大鼠的红细胞数量显著提高。在第10d红细胞压积从46±4%上升至65±6%。证实了重组腺病毒AdhEPO具有潜在的临床应用价值,可用于贫血症的基因治疗。     

Use of Physcomitrella patens actin 5′ regions for high transgene expression: importance of 5′ introns

We have isolated four actin (Act) genes from Physcomitrella patens and used their corresponding 5′ regions for recombinant expression of the human vascular endothelial growth factor (rhVEGF₁₂₁) in transiently transformed Physcomitrella protoplasts and in stable transformed lines. In the transient system, we found up to 11-fold activity of the corresponding 5′ regions as compared with that of the plant constitutive 35S promoter. Moreover, the use of an optimised expression vector in which the human VEGF signal peptide was exchanged with a plant signal peptide resulted in an additional 7-fold increase in secreted rhVEGF. We found that the 5′ introns of PpAct1, PpAct5 and PpAct7 are essential for high expression. The enhancing mechanisms of the introns, however, seem to be different since in the case of PpAct1, the expression level is stimulated only in the presence of the endogenous promoter, whereas the 5′ introns of PpAct5 and PpAct7 stimulate expression also in combination with the 35S promoter. Beyond this, the isolated 5′ regions are shown to be useful for high expression levels in transgenic moss lines with values of secreted rhVEGF up to 96 μg g^-1 dry weight.A. Weise and M. Rodriguez-Franco have contributed equally to this work.     

An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate- N -acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE : AtMurE . Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds -tagged mutant alleles of AtMurE in A. thaliana . All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens ( Pp ) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens , transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.     

The moss bioreactor

The production of recombinant proteins in moss bioreactors provides all of the benefits of molecular farming in plants but avoids many plant-specific disadvantages, such as the genetic instability of de-differentiated cells in suspension culture or the lack of containment during field production. Protein yields are in the same range as those of other cell-culture-based production systems. On top of this, the moss Physcomitrella patens is the only known plant that can be genetically modified by homologous recombination, allowing efficient targeted gene disruption. Thus, the major drawback of producing human proteins in plants, allergic reactions caused by plant-specific glycosylation, can be diminished by targeted knockout of the responsible genes in moss. Unlike all other plants, moss allows straightforward 'humanisation' of plant-derived pharmaceuticals.     

Characterization and Expression Analysis of a Glutathione Reductase Gene from Antarctic Moss Pohlia nutans

Glutathione reductase (GR) is a flavoprotein oxidoreductase and plays an important role in response to oxidative stresses in plants. A cDNA-encoding cytosolic GR [GenBank accession number GACA01029426, designated as Pohlia nutans glutathione reductase gene (PnGR)] was successfully cloned from Antarctic moss P. nutans. The full-length PnGR cDNA has 1,654 bp nucleotides with an open reading frame of 1,494 bp, encoding 497 amino acid residues. The deduced amino acid sequence of PnGR had 87.0 % identity with GR in Physcomitrella patens subsp. patens. The phylogenetic analysis showed that PnGR is clustered together with known cytosolic GR in other plants. In addition, the subcellular localization analysis by observing the transient expression of PnGR–green fluorescent protein fusion protein in Arabidopsis thaliana mesophyll protoplasts also revealed PnGR targeting to cytosol in plant cells. The expression patterns of PnGR under different abiotic stresses were determined by real-time PCR. Compared to the normal condition, the maximal mRNA accumulation of PnGR increased 3.82-fold at 4 °C, 2.92-fold at 10 °C, 4.14-fold with 200 mM NaCl, and 3.17-fold with drought stress, respectively. Together, our results suggested that the inducible PnGR might play an important role in Antarctic moss P. nutans acclimatizing to polar environment.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.