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.     

Role of sugar chains in the expression of the biological activity of human erythropoietin.

Various deglycosylated derivatives of recombinant human erythropoietin (hEPO) were prepared and used to determine the role of the sugar chains in the expression of its biological activity in vivo and in vitro. Three N-linked oligosaccharides of hEPO have been partially or fully removed to obtain N-glycan (NG) (2)-, NG(1)-, and NG(0)-hEPO carrying two, one, and no N-linked sugar chains, respectively. The preparation lacking only O-linked sugar chain O O-glycan (OG) (0)-hEPO was also used. As de-N-glycosylation proceeded, the in vivo activity of the hormone decreased drastically, and the activity of these derivatives was correlated with the number of sialic acids bound to them. On the contrary, the in vitro activity was increased by the de-N-glycosylation; NG(0)-hEPO showed a 3-fold higher specific activity than the intact hormone. This was confirmed by binding experiments of the derivatives to target cells. The in vitro activity and the affinity also correlated with the number of sialic acids bound to the deglycosylated hEPO preparations. On the other hand, OG(0)-hEPO was as active as the intact hormone in vivo and in vitro. In conclusion, the N-linked sugar chains are not required for in vitro activity but required for in vivo activity, acting as anchors for the essential terminal sialic acids. The O-linked sugar chain has no essential role in the biological activity of the hormone in vivo or in vitro.     

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.     

乳腺特异高表达人促红细胞生成素转基因奶山羊的制备

目的制备乳腺特异性高表达人促红细胞生成素（hEPO）转基因奶山羊。方法采用牛β-乳球蛋白基因（BLG）调控元件和hEPO全长编码序列基因组DNA构建真核表达载体,应用受精卵原核注射的方法制备hEPO转基因山羊。结果在原核注射获得的188头羔羊中,经Southern blot法检测有4头羊含有hEPO基因,其中3头为母羊,1头公羊于出生后20d死亡;3头转基因母羊hEPO基因的拷贝数分别为1、10、2;Western blot检测结果显示转基因羊乳中的hEPO分子质量为32kDa;MTT法检测结果表明,在泌乳10d的3只转基因羊乳汁中,每毫升乳汁中hEPO活性分别达到1.17×10^2IU、1.90×10^4IU、1.91×10^4IU。结论牛BLG能够调控hEPO基因在山羊乳腺中高表达,为实现其他药用蛋白在山羊乳腺中表达奠定了基础。     

N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.     

Facile and statistical optimization of transfection conditions for secretion of foreign proteins from insect Drosophila S2 cells using green fluorescent protein reporter

Insect Drosophila melanogaster S2 cells were developed as a plasmid-based and therefore nonlytic expression system for functional foreign proteins. Transfection is an important step to introduce foreign target DNA into cells and should be properly optimized to obtain maximum production yield. Single factor search (SFS) methodology is still generally used to determine optimal condition in a biological system. Although this method is relatively simple to perform, it has many disadvantages such as not considering interactions between several factors and not covering the entire region of the solution pool. Therefore, we approached this optimization problem statistically with response surface (RSM) and evolutionary operation (EVOP) methodologies and compared the transfection efficiencies with the traditional SFS method. We employed secreted green fluorescent protein (GFP) as a reporter for determination of optimal transfection condition and secreted human erythropoietin (hEPO) as a confirming foreign model protein. Consequently, we arrived at the best optimal transient transfection condition (1 microg of plasmid DNA, 5 microg of lipofectin, 2 x 10(6) cells of initial cell number, and 18 h of transfection duration time) through a systematic access in a series of SFS, RSM, and EVOP. The secreted hEPO yield using optimal transient transfection condition by EVOP methodology was enhanced by about 1.8-fold compared to that of traditional SFS. This optimized transient transfection condition can be used as a basis for optimal stable transfections. A linear relationship between secreted GFP fluorescence intensity and secreted hEPO concentration indicated that facile and noninvasive determination of optimal transfection conditions for expression and secretion of foreign proteins in S2 cell cultures was made possible by simple measurement of GFP fluorescence.     

Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens

A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene, which occasionally results in serious physiological disorders in the transgenic animal. In this study, we report successful production of transgenic chickens that express the human erythropoietin (hEPO) gene under the control of a tetracycline-inducible promoter. A recombinant Moloney murine leukemia virus (MoMLV)-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of unincubated chicken embryos (stage X). Out of 198 injected eggs, 15 chicks hatched after 21 days of incubation and 14 hatched chicks expressed the vector-encoded hEPO gene when fed doxycycline, a tetracycline derivative, without any significant physiological dysfunctions. The expression of hEPO reverted to the pre-induction state by removing doxycycline from the diet. The biological activity of the hEPO produced in the transgenic chickens was comparable to commercially available CHO cell-derived hEPO. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. Tetracycline-inducible expression of the hEPO gene was also confirmed in the blood and eggs of the transgenic chickens.     

Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.     

人促红细胞生成素基因和GFP共表达的输卵管特异性表达载体的构建

本实验从蛋鸡输卵管基因组DNA中扩增出约1.3kb的卵清蛋白5＇端调控区（OV）,并从质粒扩增出人促红细胞生成素（hEPO）基因组DNA。将hEPO亚克隆入pEGFP-C1载体的多克隆位点,命名为pEGFP-hEPO,然后将OV片段亚克隆入经Ase I和Vsp I双酶切的切口处,替换掉CMV启动子,经测序和酶切鉴定正确,成功构建了鸡卵清蛋白5＇端调控区调控人促红细胞生成素的共表达载体pOV-GFP-hEPO。并利用脂质体转染法转染鸡输卵管上皮细胞,用荧光倒置显微镜观测绿色荧光蛋白的表达,结果显示共表达载体pOV-GFP-hEPO能够在鸡输卵管上皮细胞定位表达。为制备生产人促红细胞生成素的鸡输卵管生物反应器奠定了基础。     

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．     

Purification of erythropoietin from human plasma samples using an immunoaffinity well plate

A method is described to isolate human erythropoietin (hEPO) from plasma using an EPO-specific immunoaffinity micro well plate (IAP). The operating conditions of the method (binding, blocking and elution) were optimised to avoid isoform discrimination and cross-contamination with other glycoproteins. The overall hEPO recovery was ca. 56% and significant clean-up for plasmatic hEPO was achieved. Polyvinylpyrrolidone (PVP) was used as a blocking reagent and elution took place at pH 11.0. Under these conditions all isoforms from recombinant human EPOs (rhEPOs) and analogues were uniformly recovered guaranteeing lack of discrimination. The resulting procedure allowed isolating erythropoietin from plasma in conditions amenable to hEPO analysis by other techniques such as SDS-PAGE or IEF. Moreover, avoiding contamination with other glycosylated material allowed the identification in human plasma samples of the non-human N-glycolyl-neuraminic acid (Neu5Gc) using HPLC-FLD. Neu5Gc is present as 1–2% of the sialic acid content in rhEPO so this approach could be used to unequivocally detect abuse of rhEPOs or analogues as part of the doping control.     

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。     

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.     

Transient expression of hepatitis B virus surface antigen (HBsAg) and human erythropoietin (hEPO) genes in milk after direct introduction into ewe

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EPO修饰增强Muller细胞对RCS大鼠视网膜变性的干预效果

目的：研究人源促红细胞生成素（hEPO）修饰的Müller（hEPO-Müller）细胞对视网膜退行性病变大鼠的干预作用。方法通过质粒转染法构建hEPO和GFP的Müller细胞稳转株（hEPO-Müller和GFP-Müller）;以体外共培养和体内细胞移植为研究体系,利用RT-PCR和冰冻切片及免疫荧光染色的方法检测hEPO-Müller对RCS大鼠视网膜退行性病变的干预作用。内核层与外核层厚度比较采用t检验。结果本实验成功构建了hEPO-Müller和GFP-Müller细胞系。将RCS大鼠的视网膜组织剥离并在体外不同条件下培养两周后测定视网膜各核层厚度发现,与对照细胞裂解液共培养组的内核层（15.94±1.77）μm和外核层（24.81±3.03）μm的厚度相比较,两核层的厚度分别在hEPO组为（23.03±3.29）μm,（33.92±7.59）μm（P〈0.05）;Müller 组为（24.81±2.02）μm,（32.15±3.03）μm（P〈0.05）;hEPO-Müller组为（32.40±8.35）μm,（40.25±3.29）μm（n=3, P〈0.01）;以hEPO-Müller组厚度增加最为显著（P〈0.05）。提示EPO和Müller细胞对视网膜变性都有干预作用且两者可以叠加。将hEPO-Müller和GFP-Müller分别移植到RCS大鼠的视网膜下腔,四周后取视网膜进行冰冻切片检测,染色结果显示,细胞移植后有更多的外核层细胞存活,且同样也是hEPO-Müller组的外核层细胞更多。此外,Müller移植并不会促进视网膜的胶质化。结论移植Müller细胞可以减缓RCS大鼠视网膜变性,而经hEPO修饰的Müller细胞对视网膜变性有更好的干预作用。因此,Müller细胞可以作为一种供体细胞兼携带hEPO等营养因子的载体用于视网膜变性的治疗。     

Purification of primate T cell growth factor to apparent homogeneity by reverse phase high pressure liquid chromatography: evidence for two highly active molecularly distinct species

T cell growth factor produced by the MLA144 gibbon ape lymphosarcoma T lymphocyte line was separated into two molecular forms of Mr 16,300 and Mr 14,300 which were purified 32,000-fold and 82,000-fold, respectively, and which in combination constitute the majority of biological activity of the starting material. The overall recovery of biological activity was 50%. This was accomplished with a series of chromatographic steps including reverse-phase high pressure liquid chromatography. The purified proteins are of comparable specific activities and each maintains the DNA synthesis of T cell growth factor-dependent human T cell lines at concentrations of less than approximately 18 pM (300 fg/ml).     

hEPO cDNA重组逆转录病毒的构建

通过DNA重组技术,将不含非编码区的hEPO cDNA片段重组到逆转录病毒质粒pLXSN, pLNCX中重组质粒转染PA317细胞后,经G418筛选,抗性克隆细胞培养上清能成功地感染NIH3T3细胞,使之在筛选培养基中形成典型的G418抗性克隆,该克隆细胞染色体中成功地整合了EPOcDNA,并且表达出有生物学活性的红细胞生成素(EPO)产物。     

Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells

We sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.