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SAVAGE, MD, United States

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.

Agency: Department of Defense | Branch: Office for Chemical and Biological Defense | Program: SBIR | Phase: Phase II | Award Amount: 727.95K | Year: 2002

"Chesapeake PERL, Inc aims to develop baculovirus-based expression in insect larvae from bench-scale to mass manufacturing. The technology platform consists of automated mass insect rearing, and inoculation combined with monitoring of protein expressionlevels in larvae via a fluorescent reporter. This allows the development of "mass customization" for recombinant protein manufacturing in insect larvae that will reduce costs by nearly an order of magnitude. "Mass customization" refers to a system whereone process can make several different customized products and vary the scale of production from one to many units. The key to mass customization is that the process remains constant irrespective of product and scale. The Phase II proposal focuses on thea) development of vectors suitable for expression of different types of proteins, b) building and testing of harvesting equipment, c) development of equipment for sorting, based on fluorescence signal, d) development of protein purification protocolsstarting with larval material, e) a flow diagram with mass balance. The anticipated end result is an economically favorable process for recombinant protein manufacturing."

Agency: Department of Defense | Branch: Office for Chemical and Biological Defense | Program: SBIR | Phase: Phase I | Award Amount: 70.00K | Year: 2003

Chesapeake PERL is a contract manufacturing company using the baculovirus expression system for mass production of proteins in lepidopterous insect larvae. PERL's automated in-line insect mass rearing and inoculation systems enables the production oflarge quantities of complex, biologically fully functional proteins. The baculovirus expression system as developed in the eighties and nineties is a versatile system for protein expression at small and intermediate quantities of protein in insect cellscultured in flasks or bioreactors. We propose a series of improvements in the system aimed at developing more convenient and rapid methodology for abundant expression of both intermediate and large quantities of functional proteins in intact, livinglepidopteran larvae. to be added

Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase II | Award Amount: 2.51M | Year: 2006

The central theme of the proposed research is improvement of the quality of product proteins manufactured in insect larvae. The approach is twofold: create the most favorable conditions for the expressed protein to mature, and create the least favorable conditions for proteolytic breakdown in the insect and through downstream processing. A number of molecular biological adaptations to baculovirus expression vectors are proposed to increase the proportion of correctly folded, matured, and biologically active protein expressed. The other objectives all deal with maintaining the quality of the produced product, in the insect as well as during downstream processing. We will undertake a biochemical characterization of the proteolytic activity in infected insects, and use fluorescent reporter genes to monitor protease load. We will also characterize the type and quantity of proteolytic activity through the steps of protein separation and purification. Protease degradation in the insect will be addressed through the use of protease inhibitors and interfering RNA.

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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