Engineering of the baculovirus expression system for optimized protein production.

Baculoviruses are arthropod-specific large circular double-stranded DNA viruses successfully used for the control of multiple insect pests. In addition to their application in pest control, baculoviruses have become a versatile and powerful eukaryotic vector for the production of large quantities of recombinant proteins for research and biomedical purposes. Since the first recombinant protein was expressed in 1983 using the baculovirus expression system (BEVS), different strategies have been developed for the generation of recombinant viruses and to increase the stability, yield, and posttranslational modifications of recombinant proteins. In this review, we summarize the main methods and elements playing a role in the BEVS emphasizing recent progresses and future developments with respect to the main aspects involved in protein production using the BEVS.

Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus.

Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients' sera in the presence of 1 µM of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients' sera were performed by adding recombinant GAD65 (0.62 nM-1.4 µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded.

Nanotoxicological Effects of SiO2 Nanoparticles on Spodoptera frugiperda Sf9 Cells.

The application of silica nanoparticles (NPs) in the biomedical field experienced a great development. The driving forces for these and future developments are the possibility to design NPs with homogeneous size and structure amenable to specific grafting. Moreover, it is possible to tune the characteristics of the NPs to meet the requirements of each specific cell and desired application. Herein, we analyzed the effect of silica NPs of various sizes and surface charge on the viability of Spodoptera frugiperda cells (Sf9 cell line) with the aim of extending the knowledge of possible toxicity of the NPs in the environment and development of new tools for insect control. Moreover, these results will also contribute to develop more effective systems for gene vectors delivery and recombinant proteins expression. Bare silica NPs of 14 nm, 380 nm and 1430 nm as well as amine-modified silica NPs of 131 nm and 448 nm were obtained by the Stöber method. The NPs were characterized by DLS and zeta potential measurements. The cell viability was assessed by the MTT test. It was observed that the 14 nm NPs possess the highest toxic effect. Indeed, after 24 h, the viability of the cells exposed to the lower concentration of NPs (0.12 mg/ml) was about 40% of the value obtained for the control cells not exposed to NPs. Moreover, the exposure to other negative charged NPs also causes a lower activity when compared with the control. Alternatively, lower concentrations of positive charged NPs (i.e.: 0.12 or 0.6 mg/ml) demonstrated to stimulate the proliferation of the cells and higher concentrations (i.e.: 7.2 mg/ml) did not present significant differences with the control. In conclusion, we have demonstrated that the NPs possess an effect that is highly influenced by the size, charge and concentration. Although, silica NPs are being used in the biomedical field, these results contribute to further understanding the risk that could be associated to nanoparticles and how these can be modified in order to meet the requirements of each desired application.

Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

Kinetic characterization of human thyroperoxidase. Normal and pathological enzyme expression in Baculovirus system: a molecular model of functional expression.

BACKGROUND: Human thyroperoxidase (hTPO) is a membrane-bound glycoprotein located at the apical membrane of the thyroid follicular cells which catalyzes iodide oxidation and organification in the thyroglobulin (TG) tyrosine residues, leading to the thyroid hormone synthesis by coupling of iodotyrosine residues. Mutations in hTPO gene are the main cause of iodine organification defects (IOD) in infants. METHODS: We investigated the functional impact of hTPO gene missense mutations previously identified in our laboratory (p.C808R, p.G387R and p.P499L). In order to obtain the whole wild-type (WT) coding sequence of hTPO, sequential cloning strategy in pGEMT vector was carried out. Then, site-directed mutagenesis was performed. WT and mutant hTPOs were cloned into the pAcGP67B transfer vector and the recombinant proteins were expressed in Baculovirus System, purified and characterized by SDS-PAGE and Western blot. Moreover, we report for the first time the kinetic constants of hTPO, of both WT and mutant enzymes. RESULTS: The functional evaluation of the recombinant hTPOs showed decreased activity in the three mutants with respect to WT. Regarding to the affinity for the substrate, the mutants showed higher Km values with respect to the WT. Additionally, the three mutants showed lower reaction efficiencies (Vmax/Km) with respect to WT hTPO. CONCLUSIONS: We optimize the expression and purification of recombinant hTPOs using the Baculovirus System and we report for the first time the kinetic characterization of hTPOs.

Recombinant protein purification using complementary peptides as affinity tags.

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.