Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 µg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding.

Cloning and expression of recombinant human GMCSF from Pichia pastoris GS115--a progressive strategy for economic production.

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is a proinflammatory cytokine and hematopoietic growth factor. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF) serves as a biotherapeutic agent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. The objective of the present study includes construction of rhGMCSF having N-terminal intein tag, expression of protein both extracellularly and intracellularly from yeast expression system followed by its purification in a single step by affinity chromatography. The soluble and biologically active rhGMCSF was obtained from Pichia pastoris GS115. About 122 g DCW/L of final yield was obtained for both cytosolic and secretory expression of Pichia GS115 strain. Purified intracellular hGMCSF was 420 mg/L with a specific activity of 2.1×108 IU/mg, and the purified extracellular recombinant protein was 360 mg/L with a specific activity of 1.9×108 IU/mg. The data presented here indicate the possibilities of exploring the economic ways of producing the rhGMCSF.

A versatile ionic strength sensitive tag from a human GM-CSF-derived linear epitope.

A 7-mer hGM-CSF-derived linear epitope (APARSPS) is herein described as a novel and small tag taking into account its particular binding affinity in native conditions that could be easily modified by increasing or lowering the ionic strength. Thus, a 3.4 or 3.8-fold binding increment was observed in high NaCl concentration when the tag was fused to IFN-α2b or when the peptide was in its native environment, respectively. The high salt concentration increased the affinity of the binding interaction and improved the APARSPS epitope binding to the paratope allowing one to design an immunoaffinity chromatography purification step in which the high ionic strength was useful to adsorb the fusion protein to the immunoaffinity matrix and the low ionic strength at pH 9 was valuable to desorb it (a 470-fold purification with a 94%-purity was attained in only one step). Also, this short tag did not affect the functionality of the fusion protein and it was able to be detected both in the natural molecule (hGM-CSF) as in the tagged one with the same high detection limit: 273pg of each protein.

Production and characterization of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica.

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

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.

Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli.

Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific activity of 0.5 x 10(7) IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 x 10(7) IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly pure 14.7 kDa hGMCSF.

Co-expression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture.

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.

Comparison of four methods for the purification and refolding of human interleukin-2-mouse granulocyte/macrophage colony-stimulating factor fusion protein.

The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2-mGM-CSF (human IL-2-mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His(6)). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2-mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7 x 10(6) and 1.1 x 10(7) i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2-mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2-mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.

Development of efficient method for purified recombinant bovine granulocyte-macrophage colony-stimulating factor production with baculovirus-silkworm gene expression system.

Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH(4))(2)SO(4), followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6-6.3 x 10(6) ED(50) mg(-1). By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.

Expression, purification, and refolding of recombinant fusion protein hIL-2/mGM-CSF.

OBJECTIVE: To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF). METHODS: SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. RESULTS: The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4 x 10(6) IU/mg and 7.1 x 10(6) IU/mg, respectively. CONCLUSION: This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.

A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli.

Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor.

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.

A cell-free expression and purification process for rapid production of protein biologics.

Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

Identification of histamine-production-increasing factor produced by stimulated RBL-2H3 rat basophilic leukemia cells as granulocyte-macrophage colony-stimulating factor.

When RBL-2H3 rat basophilic leukemia cells were stimulated by antigen or the Ca2+ ionophore A23187, the activity to increase histamine production by rat bone marrow cells in the conditioned medium increased time-dependently. To characterize the histamine-production-increasing factor (HPIF) produced by RBL-2H3 cells, the conditioned medium was collected 8 h after stimulation by A23187, and the factor was purified by three-step chromatography, the specific activity being increased by 9000-fold. The partial amino acid sequence of the peptide obtained by S. aureus V8 protease digestion was identical to the internal amino acid sequence of rat granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, GM-CSF mRNA levels in RBL-2H3 cells were increased by A23187 with a peak at 4 h. Furthermore, recombinant rat GM-CSF increased histamine production by rat bone marrow cells. These findings suggested that HPIF produced by the stimulated RBL-2H3 cells is GM-CSF. Possible significant roles of HPIF at the late phase of allergic inflammation are discussed.

Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality.

We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 degrees C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 degrees C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N- and O-glycosylation of the protein produced under both cell culture conditions using high-pH anion-exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM-CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO-K1-hGM-CSF cells without affecting product quality, representing a novel procedure for the rhGM-CSF production process.

Development of a new technique for recovery of cytokines from inflammatory sites in situ.

We attempted to recover cytokines from nasal mucosal surface following allergen challenge. Repeated lavage of nasal mucosa of seven allergic patients was done, but we failed to detect IL-1 beta in the lavage samples even in ten-fold concentrated materials. Therefore, we developed a new technique to recover cytokines using filter strips. Small filter strips were placed on nasal turbinates for 10 min at different time points after allergen challenge. The strips were air-dried, and stored. For recovery of cytokines individual strips were washed with small volumes of Hepes buffer containing 0.3% human serum albumin. Eluates were assayed for the presence of IL-1 beta and GM-CSF using commercially available ELISA. We were able to detect IL-1 beta and GM-CSF in eluates. Both cytokines were consistently detectable in the late phase allergic reaction peaking at 5 h. Nasal challenge with saline failed to detect any cytokine during the 7 h observation period. In standardization experiments known quantities of IL-1 beta and GM-CSF were applied to filter strips and the recovery ranged from 67 to 89%. Thus, we developed a simple technique of recovery of cytokines from inflammatory mucosa in situ.

Effect of a stromal cell derived hematopoietic factor on human bone marrow progenitor cells.

Conditioned media (KM-102CM and KM-103CM) obtained from two different human bone marrow stromal cell lines (KM-102 and KM-103) were analysed for their ability to stimulate human hematopoietic stem cells. Both KM-102CM and KM-103CM stimulate the formation of granulocyte-monocyte colony forming unit (CFU-C) and erythroid burst forming unit (BFU-E) colonies in the presence of erythropoietin, and also maintain the long term proliferation of stem cells in vitro. When KM-102CM and KM-103CM were fractionated by DEAE-cellulose chromatography and treated with antiserum against granulocyte-monocyte colony stimulating factor (GM-CSF) these colony stimulating activity (CSA) and burst promoting activity (BPA) contained in these media were neutralized by the antiserum and thus proved to be basically identical to GM-CSF. These results showed that the GM-CSF produced by the marrow stromal cells can maintain and proliferate the hematopoietic progenitor cells in the long term and thus gave us an evidence of one of the regulatory functions of the marrow stromal cells in hematopoiesis.