Resume

• 2013

  Post Doctoral

  Stem Cell and Regenerative Biology Department

  PI Dr. Lee Rubin

  Stem cell modeling of neurodegenerative diseases

  Harvard University

• 2008

  Montana State University-Bozeman

  Bozeman

  Montana

  As a graduate student

  I developed a system by which any gene product is packaged in high copy number within an assembling P22 bacteriophage capsid. This new

  protein-based and self-assembling nanomaterial allows for the specific targeting of cell-types with large cargos of protein based fluorescent signal (i.e. GFP or mCherry) and/or enzymes carrying out single or sequential reactions (through encapsulation of multiple enzymes). In fact

  I showed that encapsulation of unstable enzymes greatly increases their thermal stability

  increases their reaction rate

  protects them from proteases

  and makes them able to withstand lyophilization and rehydration. This work resulted in 8 publications- 3 first author and 5 contributing.

  Research Associate

  Graduate Student

  Montana State University-Bozeman

  Bozeman

  Montana

  LigoCyte is now a part of Takeda Vaccine. As part of the Process Development team

  I devised the purification strategy for our VLP-platform vaccine from baculovirus infected insect cell cultures.

  Research Associate II

  LigoCyte Pharmaceuticals

  Inc.

  Connecticut

  USA

  My research lab is interested in why proteins aggregate in neurodegenerative disease.

  Assistant Professor

  Wesleyan University

  Stem Cell and Regenerative Biology Department. \nI use adult induced pluripotent stem cells (iPSC) to model neurodegenerative disease. With this newly realized system

  I am able to make the exact tissue that is affected in the disease- motor neurons to study ALS

  dopaminergic neurons to study Parkinson's Disease

  or cortical neurons to study Alzheimer's disease.

  Harvard University

  Excellence in Teaching

  Recognition for my high scores on the end of the year student surveys.

  Harvard University Bok Center for Teaching and Learning

  Doctor of Philosophy (PhD)

  Biochemistry

  Montana State University-Bozeman

• 2002

  Bachelor of Science (B.S.)

  Cellular and Molecular Biology

  Binghamton University

• Biochemistry

  Baculovirus

  Protein Engineering

  Bacteriophage Amplification

  Cell Culture

  Insect Cell Culture

  Transmission Electron Microscopy

  Molecular Cloning

  PCR

  Nanoparticle Characterization

  Protein Purification

  TEM

  Western Blotting

  Viral Nanotechnology

  Molecular Biology

  Science

  Protein Chemistry

  Research

  Enzyme Kinetics

  Non-Natural Amino Acid Incorporation

  Genetically Programmed In Vivo Packaging of Protein Cargo and Its Controlled Release from Bacteriophage P22

  Trevor Douglas

  Peter E. Prevelige

  Benjamin Johnson

  Courtney Reichhardt

  Packed and ready to go: A scaffold protein (SP) aids the assembly of Salmonella typhimuriam bacteriophage P22 into a capsid

  with encapsulation of the SP. This natural process was exploited by using an engineered molecular system to fuse a fluorescent protein cargo to a portion of the SP

  which templated accurate spontaneous assembly. Heating of the capsids and treatment with thrombin released the SP but not the cargo.

  Genetically Programmed In Vivo Packaging of Protein Cargo and Its Controlled Release from Bacteriophage P22

  Trevor Douglas

  Peter E. Prevelige

  Amy Servid

  Biomacromolecules

  Abstract:\nRational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy-based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends toward the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the exterior of the P22 capsid. Capsids displaying a 6xHis tag appended to the CP C-terminus bind to a Ni affinity column

  and the addition of positively or negatively charged coiled coil peptides to the capsid results in association of these capsids upon mixing. Additionally

  a single cysteine appended to the CP C-terminus results in the formation of intercapsid disulfide bonds and can serve as a site for chemical modifications. Thus

  the C-terminus is a powerful location for multivalent display of peptides that facilitate nanoscale assembly and capsid modification.\n

  Location of the Bacteriophage P22 Coat Protein C-Terminus Provides Opportunities for the Design of Capsid-Based Materials

  Emily Lin

  Trevor Douglas

  Mande Holford

  The blood brain barrier (BBB) is often an insurmountable obstacle for a large number of candidate\ndrugs

  including peptides

  antibiotics

  and chemotherapeutic agents. Devising an adroit delivery\nmethod to cross the BBB is essential to unlocking widespread application of peptide therapeutics.\nPresented here is an engineered nanocontainer for delivering peptidic drugs across the BBB\nencapsulating the analgesic marine snail peptide ziconotide (Prialt®). We developed a bi-functional\nviral nanocontainer based on the Salmonella typhimurium bacteriophage P22 capsid

  genetically\nincorporating ziconotide in the interior cavity

  and chemically attaching cell penetrating HIV-Tat\npeptide on the exterior of the capsid. Virus like particles (VLPs) of P22 containing ziconotide were\nsuccessfully transported in several BBB models of rat and human brain microvascular endothelial\ncells (BMVEC) using a recyclable noncytotoxic endocytic pathway. This work demonstrates proof in\nprinciple for developing a possible alternative to intrathecal injection of ziconotide using a tunable\nVLP drug delivery nanocontainer to cross the BBB.

  Tailored Delivery of Analgesic Ziconotide Across a Blood Brain Barrier Model Using Viral Nanocontainers

  Trevor Douglas

  Peter E. Prevelige

  Abstract:\nVirus like particles

  and other naturally occurring protein containers

  have emerged as excellent building blocks for nanomaterials design and synthesis. Here we exploit a directed assembly and encapsulation approach to sequester multiple copies of a phosphotriesterase (PTE) enzyme within the capsid of bacteriophage P22. Phosphotriesterase

  from Brevundimonas diminuta

  is an intriguing enzyme as it is highly active against a wide range of harmful insecticides and nerve agents such as Soman and Sarin. However

  difficulty in expressing large quantities of the active recombinant enzyme has limited efforts to scale-up its use. Additionally

  as a mesophilic enzyme its low heat tolerance and susceptibility to proteolysis makes it a less than ideal candidate as a practical bioremediation tool. Through encapsulation of the PTE within the P22 capsid

  we demonstrate a greatly enhanced thermal tolerance of the enzyme

  maintaining 50% of its activity to 60 °C. Additionally

  the P22 capsid confers protection to the enzyme from proteases

  as well as stabilizing the enzyme against desiccation. Thus

  our engineered P22 encapsulation system greatly enhances the stability of the mesophilic phosphotriesterase and results in a robust and active nanoparticle reactor.

  Stabilizing Viral Nano-Reactors for Nerve Agent Degradation

  Trevor Douglas

  Peter E. Prevelige

  Gautam Basu

  Abstract:\nThe precise architectures of viruses and virus-like particles are proving to be highly advantageous in synthetic materials applications. Not only can these nanocontainers be harnessed as active materials

  but they can be exploited for examining the effects of in vivo “cell-like” crowding and confinement on the properties of the encapsulated cargo. Here we report the first example of intermolecular communication between two proteins coencapsulated within the capsid architecture of the bacteriophage P22. Using a genetically engineered three-protein fusion between the P22 scaffold protein

  and the FRET pair

  GFP

  and a red fluorescent protein (mCherry)

  we were able to direct the encapsulation of the genetic fusion when coexpressed with P22 coat protein. These self-assembled P22 capsids are densely packaged

  occupying more than 24% of the available volume

  and the molecular design assures a 1:1 ratio of the interacting proteins. To probe the effect of crowding and confinement on the FRET communication in this nanoenvironment

  we spaced the donor–acceptor pair with variable length flexible linkers and examined the effect on FRET inside the capsid compared to the same tethered FRET pairs free in solution. The P22 system is unique in that the capsid morphology can be altered

  without losing the encapsulated cargo

  resulting in a doubling of the capsid volume. Thus

  we have additionally examined the encapsulated fusions at two different internal concentrations. Our results indicate that FRET is sensitive to the expansion of the capsid and encapsulation enforces significant intermolecular communication

  increasing FRET by 5-fold. This P22 coencapsulation system is a promising platform for studying crowding

  enforced proximity

  and confinement effects on communication between active proteins.

  Coconfinement of Fluorescent Proteins: Spatially Enforced Communication of GFP and mCherry Encapsulated within the P22 Capsid

  My research lab is interested in understanding why proteins aggregate in neurodegenerative disease. We use human iPSC to model the unique cellular milieu that gives rise to the intra-cellular aggregates that are the hallmarks of disease.

  Alison

  LigoCyte Pharmaceuticals

  Inc.

  Harvard University

  Wesleyan University