Ralph F. Hopkins

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling

Birt–Hogg–Dubé syndrome, a hamartoma disorder characterized by benign tumors of the hair follicle, lung cysts, and renal neoplasia, is caused by germ-line mutations in the BHD(FLCN) gene, which encodes a tumor-suppressor protein, folliculin (FLCN), with unknown function. The tumor-suppressor proteins encoded by genes responsible for several other hamartoma syndromes, LKB1, TSC1/2, and PTEN, have been shown to be involved in the mammalian target of rapamycin (mTOR) signaling pathway. Here, we report the identification of the FLCN-interacting protein, FNIP1, and demonstrate its interaction with 5′ AMP-activated protein kinase (AMPK), a key molecule for energy sensing that negatively regulates mTOR activity. FNIP1 was phosphorylated by AMPK, and its phosphorylation was reduced by AMPK inhibitors, which resulted in reduced FNIP1 expression. AMPK inhibitors also reduced FLCN phosphorylation. Moreover, FLCN phosphorylation was diminished by rapamycin and amino acid starvation and facilitated by FNIP1 overexpression, suggesting that FLCN may be regulated by mTOR and AMPK signaling. Our data suggest that FLCN, mutated in Birt–Hogg–Dubé syndrome, and its interacting partner FNIP1 may be involved in energy and/or nutrient sensing through the AMPK and mTOR signaling pathways.

Filovirus-Like Particles Produced in Insect Cells: Immunogenicity and Protection in Rodents

BACKGROUND Virus-like particles (VLPs) of Ebola virus (EBOV) and Marburg virus (MARV) produced in human 293T embryonic kidney cells have been shown to be effective vaccines against filoviral infection. In this study, we explored alternative strategies for production of filovirus-like particle-based vaccines, to accelerate the development process. The goal of this work was to increase the yield of VLPs, while retaining their immunogenic properties. METHODS Ebola and Marburg VLPs (eVLPs and mVLPs, respectively) were generated by use of recombinant baculovirus constructs expressing glycoprotein, VP40 matrix protein, and nucleoprotein from coinfected insect cells. The baculovirus-derived eVLPs and mVLPs were characterized biochemically, and then the immune responses produced by the eVLPs in insect cells were studied further. RESULTS The baculovirus-derived eVLPs elicited maturation of human myeloid dendritic cells (DCs), indicating their immunogenic properties. Mice vaccinated with insect cell-derived eVLPs generated antibody and cellular responses equivalent to those vaccinated with mammalian 293T cell-derived eVLPs and were protected from EBOV challenge in a dose-dependent manner. CONCLUSION Together, these data suggest that filovirus-like particles produced by baculovirus expression systems, which are amenable to large-scale production, are highly immunogenic and are suitable as safe and effective vaccines for the prevention of filoviral infection.

A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line

A new rapid method for titrating baculovirus stocks has been developed using a novel cell line Sf-9 Easy Titer (Sf-9ET). The Sf-9ET cell line has been transfected with plasmid DNA containing the enhanced green fluorescent protein (eGFP) gene under the control of the baculovirus polyhedrin promoter. When used in the titration assay, the Sf-9ET cells turn green when they are infected with baculovirus due to the activation of the polyhedrin promoter/eGFP complex by baculovirus gene products expressed during the infection. Using a 96-well plate format end-point dilution assay, baculovirus titers can be determined in three days using a fluorescence microscope.

Detection of antibodies to Kaposi’s Sarcoma-Associated Herpesvirus: a new approach using K8.1 ELISA and a newly developed recombinant LANA ELISA

Detection of antibodies to Kaposis sarcoma-associated herpesvirus (KSHV or Human herpesvirus 8) is a topic of ongoing controversy. KSHV expresses multiple antigens and host responses are highly variable. We have previously described an algorithm for determining KSHV infection based on K8.1 ELISA and LANA immunofluorescence assay (IFA). Here we describe the development of a recombinant ELISA for LANA and an improved testing strategy using ELISAs for LANA and K8.1. We assessed mammalian and baculovirus expression systems for the production of full-length recombinant LANA. We evaluated the performance of LANA ELISAs using human serum samples from several sources including blood donors and clinical patients diagnosed with Kaposis sarcoma and compared them to LANA IFA. Both LANA ELISAs exhibited comparable sensitivity and specificity to LANA IFA but showed considerably greater reliability. The LANA ELISA can thus be used in conjunction with the previously described K8.1 ELISA to enable the highly sensitive and specific detection of antibodies to KSHV. Use of this testing strategy will provide a more accurate and reliable diagnostic assessment of KSHV status.

Purify First: rapid expression and purification of proteins from XMRV.

Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale.

Optimizing Transient Recombinant Protein Expression in Mammalian Cells

Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression.

Widening the bottleneck: increasing success in protein expression and purification.

The number of variables at play in the expression and purification of a single protein dwarf those involved in sequencing a genome. Although certain trends are apparent, there is no one-size-fits-all approach to the process of purifying proteins. Thus, whereas numerous genome sequencing projects are providing an overwhelming number of interesting open reading frames for structural biologists to study, fully realizing the potential of this resource is still only a distant hope. We will discuss several current approaches to high throughput expression and purification as well as strategies that have served us well to quickly identify lead protein expression constructs in the context of a core service protein expression and purification laboratory. The use of the baculovirus expression vector system and implementation of a purification screening method will be emphasized.