Production, processing and partial purification of functional G protein beta gamma subunits in baculovirus-infected insect cells.
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ABSTRACT:
SUBMITTER: Robishaw JD
PROVIDER: S-EPMC1132956 | biostudies-other | 1992 Sep
REPOSITORIES: biostudies-other
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Project description:The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc.
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Project description:Diacylglycerol kinases (DGKs) are lipid kinases that modulate the levels of lipid second messengers, diacylglycerol and phosphatidic acid. Recently, increasing attention has been paid to its ? isozyme (DGK?) as a potential target for cancer immunotherapy. DGK? consists of the N-terminal regulatory domains including EF-hand motifs and C1 domains, and the C-terminal catalytic domain (DGK?-CD). To date, however, no structures of mammalian DGKs including their CDs have yet been reported, impeding our understanding on the catalytic mechanism of DGKs and the rational structure-based drug design. Here we attempted to produce DGK?-CD or a full-length DGK? using bacterial and baculovirus-insect cell expression system for structural studies. While several DGK?-CD constructs produced using both bacterial and insect cells formed insoluble or soluble aggregates, the full-length DGK? expressed in insect cells remained soluble and was purified to near homogeneity as a monomer with yields (1.3 mg/mL per one L cell culture) feasible for protein crystallization. Following enzymatic characterization showed that the purified DGK? is in fully functional state. We further demonstrated that the purified enzyme could be concentrated without any significant aggregation, and characterized its secondary structure by circular dichroism. Taken together, these results suggest that the presence of N-terminal regulatory domains suppress protein aggregation likely via their intramolecular interactions with DGK?-CD, and demonstrate that the baculovirus-insect cell expression of the full-length form of DGK?, not DGK?-CD alone, represents a promising approach to produce protein sample for structural studies of DGK?. Thus, our study will encourage future efforts to determine the crystal structure of DGK, which has not been determined since it was first identified in 1959.
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Project description:Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFN?) was expressed in insect larvae that constitute important agronomic plagues. rFeIFN? accumulated in the hemolymph of Spodoptera frugiperda larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFN? was recovered after purification with a specific activity of 1?×?106 IU mg-1. By this method, we obtained 8.9?×?104 IU of purified rFeIFN? per larva. The product was biologically active in vitro, with an antiviral activity of 9.5?×?104 IU mL-1, as well as a potent antitumor activity comparable to that of the commercial FeIFN?. The glycosylation of rFeIFN? was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFN? production in laboratory research or veterinary medicine applications.
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Project description:The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.
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Project description:Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanum tuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal and antibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state using bacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility, misfolding or low yields.In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insect cells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinant peptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. An optimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gave rise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stable transgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinant antimicrobial peptides expression in other heterologous systems.The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide in baculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploited for its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of the baculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functional analysis and antibody production, as an important step to both elucidate their accurate physiological role and to deepen the study of their biotechnological uses.
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Project description:The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains.
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Project description:Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core ?1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral vector can be used to solve the immunogenic core ?1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core ?1,6-fucosylation.
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