Production of biologically active human myelocytokines in plants

An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.     

启动子Ptac与PsbA在鱼腥藻7120中表达hG-CSF的效率比较

为了比较外源性启动子Ptac与内源性启动子PsbA在鱼腥藻7120中表达外源基因时的效率,构建了分别含Ptac和PsbA两种启动子的穿梭表达载体pRL-PsbA-GCSF、pRL-Tac-GCSF;利用三亲结合转移法转化鱼腥藻7120,利用抗生素筛选,通过质粒提取和PCR方法鉴定,获得了分别由2种启动子驱动表达hG-CSF的转基因蓝藻,转基因藻中目的基因以质粒形式存在;利用半定量RT-PCR方法对2种转基因藻的hG-CSF转录水平进行比较,发现PsbA启动子驱动效率与Ptac启动子没有明显差异;利用ELISA方法比较hG-CSF蛋白表达量,发现PsbA启动的蓝藻中hG-CSF表达量是Ptac诱导条件下表达量的1.17倍。     

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l⁻¹ during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as ~64 g dry cell wt l⁻¹, 223 mg hG-CSF g⁻¹ dry cell wt and 775 mg hG-CSF l⁻¹ h⁻¹, respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.     

Human granulocyte colony stimulating factor (hG-CSF): cloning,overexpression, purification and characterization

Background  

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.     

A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures

Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice alpha-amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was confirmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was confirmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF.     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。     

High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli

Secretory production of human granulocyte colony-stimulating factor fusion protein (hG-CSF) by fed-batch culture of Escherichia coli was investigated in both 2.5-L and 30-L fermentors. To develop a fed-batch culture condition that allows efficient production of hG-CSF, different feeding strategies including pH-stat, exponential and constant feeding were examined. Among these, the constant feeding strategy (0.228 g glucose2min^-1) and the exponential feeding that supports a low specific growth rate (µ=0.116 h^-1) resulted in the best hG-CSF production. Under these conditions, 4.4 g2L^-1 of hG-CSF was produced. The effect of induction time on the protein production was also investigated. For the fed-batch cultures carried out with the pH-stat and exponential feeding strategies, induction at higher cell density (late-exponential phase) resulted in more hG-CSF production compared with induction at lower cell density (early to mid-exponential phase). The constant feeding strategy that supported best hG-CSF production was applied to the scale-up production of hG-CSF in 30 L of fermentor. The maximum dry cell weight and hG-CSF concentration of 51.7 and 4.2 g2L^-1, respectively, was obtained.     

Improved process for production of recombinant yeast-derived monomeric human G-CSF

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secretion efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents. Received: 2 March 1999 / Received revision: 16 April 1999 / Accepted: 23 April 1999     

Proteolysis and synthetic strategy of human G-CSF in Escherichia coli BL21(DE3)

We report for the first time that the C-terminal region of hG-CSF suffers from proteolytic degradation when human granulocyte colony-stimulating factor (hG-CSF) is directly expressed in Escherichia coli BL21(DE3). It is believed that the rapid proteolysis occurs at the C-terminus of hG-CSF that is very easily exposed to E. coli protease(s) during a short period following protein synthesis and prior to completion of the formation of the inclusion body. The recombinant hG-CSF that is expressed with an N-terminal fusion partner is effectively protected from the proteolysis. It seems that since the N-terminus of hG-CSF is located very close to the C-terminus, the presence of the N-terminal fusion partner masks the C-terminal region of hG-CSF and protects it from proteolytic degradation by E. coli protease(s). Furthermore, the solubility of hG-CSF markedly increased in E. coli cytoplasm when a stress-responsive and aggregation-resistant protein, i.e. aspartate carbamoyl-transferase catalytic chain (PyrB) was used as a novel N-terminal fusion partner proteins.     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.     

Construction and characterization of a novel maltose inducible expression vector in Bacillus subtilis

A maltose-inducible expression vector in Bacillus subtilis has been developed and characterized. The vector permitted β-galactosidase expression at a high level (maximum activity, 8.16 U/ml) when induced and its expression was markedly repressed by glucose. Using this vector, we successfully expressed the other two genes, bioA and vgb. This thus provided a potential expression system for cloned genes in B. subtilis.     

Enhanced Heterologous Expression of Biologically Active Human Granulocyte Colony Stimulating Factor in Transgenic Tobacco BY-2 Cells by Localization to Endoplasmic Reticulum

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.     

A System for Dual Protein Expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

Dynamics of Recombinant hG-CSF in Transgenic Goat: Preliminary Study in the Founder during Hormonally Induced Lactation

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.     

A novel binary expression vector for production of human IL-10 in Escherichia coli and Bifidobacterium longum

A novel expression vector (pLR) driven by hup promoter and Bifidobacterium β-galactosidase signal peptide was constructed. The pLR vector was used for the expression of the optimized human IL-10 synthetic gene in Escherichia coli and Bifidobacterium longum. In both microorganisms, rhIL-10 was in a soluble form in total extract cells. The recombinant hIL-10 was partially processed in E. coli, whereas in Bifidobacterium all rhIL-10 was found in the mature form.