Aptamers improve the expression of a human granulocyte-macrophage colony stimulating factor in transgenicArabidopsis thaliana seeds

Domain IV nucleotides of human 7SL RNA comprise an approximately 50-nucleotide region with a highly conserved secondary structure consisting of two internal loops. We inserted these into a modified 3’-untranslated region of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene. The expression vector, under the control of a tissue-specific promoter derived from the soybean α’ subunit of β-conglycinin, was introduced intoArabidopsis thaliana byAgrohacterium-mediated transformation. Expression of the recombinant hGM-CSF significantly improved, accounting for as much as 0.049% of the total soluble protein in theArabidopsis siliques. Western blots showed that the molecular weight of this recombinant hGM-CSF was approximately 21 kD. In addition, TF-1 cell proliferation data demonstrated that the recombinant hGM-CSF was biologically active. Our results provide evidence that aptamers can strongly enhance gene expression.     

Quick method of multimeric protein production for biologically active substances such as human GM-CSF (hGM-CSF)

The C-terminal fragment of C4b-binding protein (C4BP)-based multimerizing system was applied to hGM-CSF to induce dendritic cells (DCs) from peripheral blood monocytes (PBMCs), to see whether the C4BP could stimulate immature DCs, since DCs, equipped with pattern recognition receptors such as toll-like receptors (TLRs), are hypersensitive to various immunologically active molecules like LPS. hGM-CSF gene was merged to the 3′-terminal region of the C4BPα-chain gene, and the transfected human 293FT cells produced sufficient amount of octameric hGM-CSF, which resulted in iDCs with the same phenotype and the same response to a TRL4 ligand, LPS and a TLR3 ligand, poly I:C, as those induced with authentic monomeric hGM-CSF. These results suggest that the C4BP-based multimerizing system could facilitate the design of self-associating multimeric recombinant proteins without stimulating iDCs, which might be seen with the other multimerizing systems such as that using Fc fragment of IgM.     

Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.     

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.     

Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.     

A Simplified Method for the Efficient Refolding and Purification of Recombinant Human GM-CSF

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.     

Development of an α-amylase reporter system for efficient screening of clones with highly expressed heterologous protein in Hansenula polymorpha

To check feasibility and effectiveness of the α-amylase reporter system, two vectors were designed and tested using hepatitis B virus surface antigen (HBsAg) and Homo sapiens granulocyte-macrophage colony stimulating factor 2 (hGM-CSF2) as a model. By integrating the vector containing two independent cassettes into the same genome locus, high-producing clones of HBsAg (or hGM-CSF2) were screened using the α-amylase as a reporter. Results show there was a positive correlation (Correlation coefficient, R ² > 0.95) between the yield of recombinant proteins and the α-amylase activity of corresponding transformants, which was independent of the gene dosage.     

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.     

Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent

A method for the production and purification of biologically active recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), expressed in Escherichia coli, has been developed. Washing of an inclusion body fraction which allows the removal of numerous ballast proteins and on-column refolding of rhGM-CSF are specific characteristics of the method. The refolding efficiency reached 70%; the purity of the preparation constituted 95%. The biological activity was similar to those of other recombinant hGM-CSF analogs.     

Improved Production of the Human Granulocyte-Macrophage Colony Stimulating Factor in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds Using a Dual Sorting Signal Peptide

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) containing either an endoplasmic reticulum (ER) retention signal or a phaseolin vacuolar sorting signal peptide was expressed in Arabidopsis thaliana under the control of a tissue-specific promoter, derived from the soybean α′ subunit of β-conglycinin. No significant differences in recombinant hGM-CSF (rhGM-CSF) accumulation were detected between transgenic plants carrying either one of the two signal peptides. Hybrid seed from crosses between single-copy transformants tailed with the ER retention signal tetrapeptide and single-copy transformed plants tagged with a phaseolin four carboxy-terminal residues showed gene additive effects. The highest expression level of rhGM-CSF was 0.05% of total soluble protein of immature siliques, indicating that the two signal peptides functioned independently in the protein-sorting pathway. Additionally, TF-1 cell proliferation data demonstrated that rhGM-CSF was biologically active.     

Secretory production of hGM-CSF with a high specific biological activity by transgenic plant cell suspension culture

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM-CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 μg/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 μg/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation fromE. coli.     

Expression of the human granulocyte–macrophage colony stimulating factor (hGM-CSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice

Expression of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected “protective or enhancer effect” from the MAR element on the hGM-CSF gene expression was not observed.     

优化mRNA翻译起始区提高人粒－巨噬细胞集落刺激因子在E．coli中的表达

人粒－巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）是一种重要的造血生长因子．利用基因重组技术构建两个ｈＧＭ－ＣＳＦ的Ｅ．ｃｏｌｉ表达菌株,一个为在不改变氨基酸顺序的前提下,对ｍＲＮＡ翻译起始区核苷酸顺序进行优化突变（ｈＧＭ－ＣＳＦ（Ｍ））,另一个为未突变的对照（ｈＧＭ－ＣＳＦ（Ｎ））．经酶切电泳、ＤＮＡ测序、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ等分析鉴定,证明两者均能表达特异性的１４．６ｋＤｈＧＭ－ＣＳＦ,但ｈＧＭ－ＣＳＦ（Ｍ）的表达水平较ｈＧＭ－ＣＳＦ（Ｎ）提高了１．２６倍,占菌体总蛋白的１６．９％．ｍＲＮＡ翻译起始区二级结构预测分析表明,优化突变后生成自由能ΔＧ从原来的－１０．２提高至－９．４Ｋｃａｌ,ＡＵＧ从部分配对状态变为非配对状态．     

Modification of the N-Terminal Nucleotide Sequence of Mature hGM-CSF Results in High Expression in the Foreign Host, Anabaena sp. Strain PCC 7120

Cloning and high foreign expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene were achieved in Anabaena sp. strain PCC 7120 cells. To promote high expression of hGM-CSF in cyanobacterial cells, PCR primers were designed to modify the N-terminal cDNA sequence of mature hGM-CSF, including a GC rich region and some discriminating against codons according to the degeneracy codon rules, selecting for prokaryotic usage codons. The PCR product encoding the modified hGM-CSF was inserted downstream of the promoter, PpsbA of the shuttle vector pRL439, then ligated with pDC-08 to generate the shuttle expression plasmid, pDC-GM1. The resulting shuttle expression plasmid was transferred into the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120 using the tri-parental conjugation transfer method. The results of PCR amplification of wild type and transgenic cells indicated that the hGM-CSF gene was successfully cloned into Anabaena sp. strain PCC 7120 cells. Western blot analysis showed that the protein expression of modified hGM-CSF in transgenic cells harboring pDC-GM1 was 136% higher than that of non-modified hGM-CSF in transgenic cells harboring pDC-GM0. Additionally, there were similar rate of growth and content of Chl a as compared to controls, suggesting that foreign hGM-CSF did not impair the photosynthetic activity of host cells. Taken together, the results indicate that modification of the N-terminal nucleotide sequence of mature hGM-CSF results in high expression in the transgenic cells.     

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.     

Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF

The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.     

非转座子载体介导的稳定转化家蚕BmN细胞表达人粒细胞-巨噬细胞集落刺激因子

为了建立非转座子载体介导的持续表达外源基因的转化家蚕BmN细胞系,将家蚕核型多角体病毒极早期基因(ie-1)启动子控制的人粒细胞-巨噬细胞集落刺激因子(hGM-CSF)基因的表达盒克隆至pIZT/V5-His,获得重组载体pIZT-IE-hGM-CSF,该载体转染家蚕BmN细胞后,通过博莱霉素(Zeocin)筛选获得了稳定转化细胞系IE-hGM-CSF。转基因细胞基因组经PCR鉴定,成功检测到ie-hGM-CSF,Western blotting分析结果显示转化细胞表达的重组hGM-CSF的大小为22kDa,ELISA检测结果显示hGM-CSF在转化细胞系里的表达水平大约为2814.7pg/106个细胞。     

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.