SAVAGE, MD, United States
SAVAGE, MD, United States
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Otto T.C.,U.S. Army | Kasten S.A.,U.S. Army | Kovaleva E.,Chesapeake Perl | Liu Z.,Chesapeake Perl | And 7 more authors.
Chemico-Biological Interactions | Year: 2010

Human serum paraoxonase-1 (HuPON1) is difficult to either purify from plasma or functionally express in high yield from recombinant sources. Here, we describe the characterization of functional HuPON1 expressed and purified from Trichoplusia ni (T. ni) larvae infected with an orally active form of baculovirus. SDS-PAGE and anti-HuPON1 Western blot analyses yielded only three bands of approximately 41, 42, and 44. kDa. MALDI-TOF confirmed the identity of each of these bands as HuPON1 with greater than 95% confidence. These isoforms result from differential glycosylation of the enzyme as indicated by peptide mapping, mass analysis, and PNGase F deglycosylation experiments. Recombinant insect-produced HuPON1 hydrolyzed phenyl acetate, paraoxon, and the nerve agents GF, VX, and VR. The enzyme had dramatic stereoselectivity for the P+ isomers of VX and VR. T. ni larvae expressing HuPON1 were remarkably resistant to the pesticide chlorpyrifos. Together, these results demonstrate that the caterpillar of the T. ni moth can be used as an expression system to produce large quantities of functional recombinant HuPON1. Insect production of HuPON1 may provide a source for both in vitro enzymatic and crystallographic studies and in vivo stability and anti-nerve agent efficacy testing. © 2010.

Buchman G.W.,Chesapeake Perl | Cohen M.E.,University of Pennsylvania | Xiao Y.,University of Pennsylvania | Richardson-Harman N.,Alpha StatConsult LLC | And 6 more authors.
Vaccine | Year: 2010

Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination. © 2010 Elsevier Ltd.

Coy M.R.,University of Florida | Salem T.Z.,University of Florida | Denton J.S.,University of Florida | Kovaleva E.S.,Chesapeake Perl | And 7 more authors.
Insect Biochemistry and Molecular Biology | Year: 2010

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion. © 2010 Elsevier Ltd.

Scharf M.E.,University of Florida | Kovaleva E.S.,Chesapeake Perl | Jadhao S.,University of Florida | Campbell J.H.,Chesapeake Perl | And 2 more authors.
Insect Biochemistry and Molecular Biology | Year: 2010

This research focused on digestive beta-glucosidases from glycosyl hydrolase family (GHF) 1 from the gut of the lower termite Reticulitermes flavipes. In preceding studies on R. flavipes, we characterized beta-glucosidase activity across the gut and its inhibition by carbohydrate-based inhibitors, and subsequently we identified two partial beta-glucosidase cDNA sequences from a host gut cDNA library. Here, we report on the full-length cDNA sequence for one of the R. flavipes beta-glucosidases (RfBGluc-1), the expression of its mRNA in the salivary gland and foregut, the production of recombinant protein using a baculovirus-insect expression system, optimal recombinant substrate specificity profiles and parameters, and significant inhibition by the established beta-glucosidase inhibitor cellobioimidazole. We also report the partial cDNA sequence for a second gut beta-glucosidase (RfBGluc-2), and show that like RfBGluc-1 its mRNA is localized mainly in the salivary gland. Other results for RfBGluc-1 showing activity against laminaribose, a component of microbial cell walls, suggest that RfBGluc-1 may serve dual functions in cellulose digestion and immunity. These findings provide important information that will enable the testing of hypotheses related to collaborative host-symbiont lignocellulose digestion, and that contributes to the development of next-generation termiticides and novel biocatalyst cocktails for use in biomass-to-bioethanol applications. © 2010 Elsevier Ltd.

Zhou X.,University of Florida | Kovaleva E.S.,Chesapeake Perl | Wu-Scharf D.,University of Florida | Campbell J.H.,Chesapeake Perl | And 3 more authors.
Archives of Insect Biochemistry and Physiology | Year: 2010

Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli-expressed enzyme. Km and Vmax values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056 μmol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5-7.5 and 50-60°C, is inhibited by EDTA, and displays enhanced activity up to 70°C in the presence of CaCl2. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others. © 2010 Wiley Periodicals, Inc.

Dolinska M.B.,U.S. National Institutes of Health | Kovaleva E.,Chesapeake Perl | Backlund P.,U.S. National Institutes of Health | Wingfield P.T.,U.S. National Institutes of Health | And 2 more authors.
PLoS ONE | Year: 2014

Background: Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings: The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance: The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

Sethi A.,Purdue University | Kovaleva E.S.,Chesapeake Perl | Slack J.M.,Chesapeake Perl | Brown S.,Chesapeake Perl | And 2 more authors.
Archives of Insect Biochemistry and Physiology | Year: 2013

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7-3, GHF7-5, and GHF7-6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full-length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus-insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7-3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β-glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell-1), beta-glucosidase (β-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and β-glu. Finally, GHF7-3, Cell-1, and β-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β-glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7-3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases. © 2013 Wiley Periodicals, Inc.

Sethi A.,Purdue University | Slack J.M.,Chesapeake Perl | Kovaleva E.S.,Chesapeake Perl | Buchman G.W.,Chesapeake Perl | Scharf M.E.,Purdue University
Insect Biochemistry and Molecular Biology | Year: 2013

Lignin is a component of plant biomass that presents a significant obstacle to biofuel production. It is composed of a highly stable phenylpropanoid matrix that upon degradation, releases toxic metabolites. Termites have specialized digestive systems that overcome the lignin barrier in wood lignocellulose to efficiently release fermentable simple sugars; however, how termites specifically degrade lignin and tolerate its toxic byproducts remains unknown. Here, using the termite Reticulitermes flavipes and its symbiotic (protozoan) gut fauna as a model system, we used high throughput Roche 454-titanium pyrosequencing and proteomics approaches to (i) experimentally compare the effects of diets containing varying degrees of lignin complexity on host-symbiont digestome composition, (ii) deeply sample host and symbiont lignocellulase diversity, and (iii) identify promising lignocellulase candidates for functional characterization. In addition to revealing over 9500 differentially expressed transcripts related to a wide range of physiological processes, our findings reveal two detoxification enzyme families not generally considered in connection with lignocellulose digestion: aldo-keto reductases and catalases. Recombinant versions of two host enzymes from these enzyme families, which apparently play no roles in cellulose or hemicellulose digestion, significantly enhance lignocellulose saccharification by cocktails of host and symbiont cellulases. These hypothesis-driven results provide important new insights into (i) dietary lignin as a xenobiotic challenge, (ii) the complex mechanisms used by termites to cope with their lignin-rich diets, and (iii) novel lignin-targeted enzymatic approaches to enhance biofuel and biomaterial production. © 2012 Elsevier Ltd.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 157.91K | Year: 2012

DESCRIPTION (provided by applicant): Project Summary: Lung cancer is the leading cause of cancer death in the United States and is responsible for more deaths each year than breast, prostate, colon, hepatic, renal and skin cancers combined. Viewed in economic terms, in the United States, the value of life lost from lung cancer deaths in the year 2000 was more than 240 billion, which is estimated to rise to more than 433 billion in 2020. Because of the immense health care and economic burden imposed by lung cancer, new therapy strategies that improve patient outcomes will lead to very a significant impact. Collaborators at UCLA (Laboratory of Lenny Rome) have identified and characterized a unique ribonucleoprotein nanoparticle structure that is highly stable, and is found ubiquitously in all higher eukaryotes. Major Vault Protein (MVP) is the core nanoparticle component, and it is readily engineered to permit attachment of other agents, including promising cancer therapeutics like chemokine ligand 21 (CCL21). CCL21 is a lymphoid chemokine that is chemoattractant for mature dendritic cells (DCs), naive and memory T cells. Preclinical studies have demonstrated that intratumoral administration of CCL21 gene-modified dendritic cells led to tumor eradication. Vaults have been expressed at Chesapeake PERL, Inc. (C-PERL) to very high levels using the PERLXpress protein expression platform. The unique and powerful system uses recombinant baculovirus expression in whole insects in an automated platform to generate high protein yields cost effectively. Preliminary data shows that MVP is readily expressed and correctly assembled to form nanoparticles. Further, packaging of a model protein (pCherry) has been demonstrated. . We expect that continued development will showthat CCL21-Vaults can be readily prepared in high quality by expression, and packaging of the constituent proteins, followed by stringent purification methods to generate high yields of high quality nanocapsule. We hypothesize that intratumoral administration of recombinant CCL21-vaults derived from baculovirus infection of whole insects will circumvent autologous DC preparation, minimize batch to batch variability and allow for comparability and standardization so that the particle can be used as an off-the-shelf reagent for advanced NSCLC. Specific Aim 1: Engineer, express and purify Vault-CCL21 nanocapsule protein complexes for initial biochemical characterization using standard protein analytical tools as well as electron microscopy. Continue purification development to optimize yield and ensure highest quality and purity. Specific Aim 2: Perform in vitro analyses, including chemotactic and related assays to assess and compare the biochemical profile for CCL21-vault and compare to previously tested CCL21-vault expressed in SF9 cells. PUBLIC HEALTH RELEVANCE: This project is relevant to treatment of common causes of death and disability due to lung cancer. The research will support continued development of a candidate therapy using a recombinant version of naturally occurring human vault protein in combination with CCL21 antitumor agent. The combination technologies may result in a drug that targets lung cancer, demonstrating a large scale, low cost, reproducible therapy. This is a potentially disruptive approach that can revolutionize the lung cancer field, and may be applied to other cancers.

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