Efficient Plant-Based Production of Chicken Interleukin-12 Yields a Strong Immunostimulatory Cytokine

Medrano, Giuliana; Dolan, Maureen C.; Stephens, Nathan T.; McMickle, Anthony; Erf, Gisela F.; Radin, David N.; Cramer, Carole L.

doi:10.1089/jir.2009.0040

Cited by 14 publications

(12 citation statements)

References 36 publications

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“…To generate a DNA fragment, Kozak/chIL-12, the chIL-12-encoding region containing the p40 and p35 subunits was constructed into a heterodimeric single chain as described. 9 Forward (fwd) and reverse (rev) pchIL-12 primers were used to amplify the chIL-12-encoding region from the total DNA templates prepared from rFPV-infected DF1 cells by using PCR as described previously. 9 The PCR products were treated with Kpn I and Xho I ( Figure 1A).…”

Section: Construction Of Plasmid Insertion Vectorsmentioning

confidence: 99%

“…9 Forward (fwd) and reverse (rev) pchIL-12 primers were used to amplify the chIL-12-encoding region from the total DNA templates prepared from rFPV-infected DF1 cells by using PCR as described previously. 9 The PCR products were treated with Kpn I and Xho I ( Figure 1A). To generate an insertion vector that contained a selection marker (eGFP) gene for purifying the stable cell line and analyzing protein synthesis, peGFP (fwd) and (rev) primers derived from a peGFP plasmid (Clontech) were used to amplify the eGFP gene through PCR.…”

Section: Construction Of Plasmid Insertion Vectorsmentioning

confidence: 99%

“…Fusion proteins were detected by conducting western blotting by using an antibody recognizing eGFP (GeneTex), FLAG (GeneTex), or chIL-12 expressed by E. coli (echIL-12) as the probe. 9 To further determine whether the expressed protein was a fusion protein, the protein-containing band with the expected size after SDS-PAGE was cut for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (ABI Voyager-DEPRO). The peptide masses obtained were searched against a comprehensive nonredundant protein sequence database (NCBInr) by using the Mascot program for protein identification.…”

Section: Construction Of Plasmid Insertion Vectorsmentioning

confidence: 99%

“…In brief, peGFP (fwd) and peGFP (rev) primers (Table 1) were used to amplify the eGFP-encoding region by using the cDNA sequence of the eGFP in a peGFP plasmid (Clontech, USA) as the template. 9 The PCR products were cloned into pET28a to generate pET28a/eGFP. The procedures for eGFP-FLAG expression, purification, and identification were executed as described.…”

Section: Determination Of the Total Fusion Protein Concentrationmentioning

confidence: 99%

“…6 As observed in mammalian IL-12 (molecular size, 74 kDa), 3,7 heterodimerization of p40 and p35 of chIL-12 is essential for its functioning. In addition to the expression of chIL-12 in insect cells and COS cells for its characterization, 6 the synthesis of functionally active chIL-12 in Escherichia coli, 8 plants, 9 and recombinant fowlpox virus (rFPV) 10 has been described. Thus, recombinant chIL-12 (rchIL-12) appears to be a promising vaccine adjuvant.…”

Section: Introductionmentioning

confidence: 99%

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Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

Chen

Shen

et al. 2015

Biotechnology Progress

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The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12.

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Section: Construction Of Plasmid Insertion Vectorsmentioning

confidence: 99%

Section: Construction Of Plasmid Insertion Vectorsmentioning

confidence: 99%

Section: Construction Of Plasmid Insertion Vectorsmentioning

confidence: 99%

Section: Determination Of the Total Fusion Protein Concentrationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

Chen

Shen

et al. 2015

Biotechnology Progress

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Quality Assessment of Recombinant Proteins Produced in Plants

Medrano

Dolan

Condori

et al. 2011

Recombinant Gene Expression

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Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy presented using the ChIL-12(HIS) example should provide the basis of standard procedures for assessing the quality of other plant-based protein products and manufacturing systems.

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Tools of the Trade: Developing Antibody-Based Detection Capabilities for Recombinant Proteins

Dolan

Medrano

McMickle

et al. 2011

Recombinant Gene Expression

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Protein-specific antibodies serve as critical tools for detection, quantification, and characterization of recombinant proteins. Perhaps the most important and widely used antibody-based procedures for recombinant protein applications are Western immunoblotting and enzyme-linked immunosorbent assays (ELISAs). These analyses require well-characterized, sensitive, and high-affinity antibodies that specifically and selectively recognize the recombinant target protein in the native or denatured form. Although the number of commercially available antibodies is quite substantial and rapidly growing, the appropriate antibody tools for many applications currently do not exist. In this chapter, strategies to develop and characterize both polyclonal and monoclonal antibodies directed against a specific protein of interest are discussed. Experimental strategies and methods are presented for producing and selecting the best antibodies and optimizing protocols for Western analyses, ELISAs, and other applications. Once antibody and procedure optimization is completed to ensure specificity, sensitivity, accuracy, and reliability, these immune-based approaches can now serve as powerful and enabling tools in the characterization, detection and diagnostics, structure/function analysis, and quality assessment of recombinant proteins.

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Efficient Plant-Based Production of Chicken Interleukin-12 Yields a Strong Immunostimulatory Cytokine

Cited by 14 publications

References 36 publications

Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

Production and characterization of active recombinant interleukin‐12/eGFP fusion protein in stably‐transfected DF1 chicken cells

Quality Assessment of Recombinant Proteins Produced in Plants

Tools of the Trade: Developing Antibody-Based Detection Capabilities for Recombinant Proteins

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