Scott A. Banta - US grants

[unreadable] DESCRIPTION (provided by applicant): The delivery of materials across the Blood Brain Barrier (BBB) to specific cellular targets is a critical challenge for the study and treatment of diseases and disorders of the brain. New methods for targeted brain delivery will dramatically improve the efficacy of both new and existing therapeutics. We propose to develop a collection of safe, effective, and widely usable peptide-based carriers that are able to deliver exogenous agents across the BBB directly to specific cell populations in the central nervous system (CNS). Recently, Cell Penetrating Peptides (CPPs) have been discovered that are capable of delivering cargos across biological membranes, including the BBB. But their therapeutic applications are limited, as they lack cell and tissue-type specificity. Homing peptides have been identified that target specific cell types, but their application is hampered by an inability to deliver cargos across membranes. In this proposal, we will use Directed Evolution to engineer novel Specific Cell Penetrating Peptides (SCPPs) that traverse the BBB and deliver materials to specific cell types. Our goal will be accomplished using a novel plasmid display system which can be used to efficiently identify SCPPs with specific cell-type penetrating activities. The plasmid display system provides a linkage between the SCPP sequence and the DNA sequence that encodes it. And, the associated plasmid serves as a cargo that can be used as a functional marker to indicate cellular penetration. Large randomized plasmid display libraries will be screened using an in vitro model of the BBB consisting of organotypic brain slice cultures grown on a confluent layer of primary endothelial cells. Fluorescence activated cell sorting (FACS) will be used to isolate specific cell types that were penetrated by the SCPPs, and the SCPPs will be identified through DNA sequencing of the attached plasmid cargo. SCPPs that are able to penetrate the BBB in vitro will be screened in vivo using an anesthetized rat. The in vivo screening will ensure that the SCPPs possess highly specific brain targeting activities and that non- specific SCPPs are eliminated from the library. The novel SCPPs engineered through this Directed Evolution approach should find immediate utility in neuroscience and behavioral research, as well as the treatment of neurodegenerative diseases. The Blood Brain Barrier (BBB) restricts the access of most materials to the central nervous system (CNS). New peptide-based carriers engineered to deliver exogenous agents across the BBB to specific cell-types in the brain will open new avenues of neuroscience and behavioral research, and they will enable new methods for the targeted delivery of therapeutic material for the treatment of various diseases of the brain. [unreadable] [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)

This award by the Biomaterials program in the Division of Materials Research to Columbia University is study the use of molecular engineering to create novel bifunctional enzymes that can self-assemble into hydrogels and support an intra-gel metabolic pathway that can oxidize methanol through two steps to formic acid. Rationally designed alpha-helical appendages will be genetically fused to the horse liver alcohol dehydrogenase homodimer enzyme and to the human liver mitochondrial aldehyde dehydrogenase homotetramer enzyme. The effects of these mutations on the performance of the enzymes will be assessed, and then the enzymes will be combined to form mixed self-assembling hydrogels that will oxidize methanol to formic acid while concomitantly producing NADH from NAD+ a cofactor required for the reaction. This will be the first proof of principle of a self-assembling bioactive biomaterial that can support a simple intra-gel metabolic network. This material could be applied to an electrode and used as an anode in an enzymatic biofuel cell using methanol as a fuel source.

Protein engineering has been used to improve globular proteins such as enzymes, and structural proteins such as the elastin-like peptides. This research project is designing and creating new proteins with both globular and structural domains to form bioactive biomaterials. Earlier studies by the researcher already modified proteins to self-assemble into enzymatic hydrogels, and in this proposal the investigators will create a new material that contains two active enzymes which can function together to form a simple metabolic pathway. The enzymes will be able to oxidize methanol first to formaldehyde and then the formaldehyde will be oxidized to formic acid. Each step will generate NADH which can be used in a power generating format such as an enzymatic biofuel cell. The creation of novel protein constructs with both useful enzymatic and materials properties is a new area in the field of protein engineering, and this approach will be very useful in the design and construction of new bioelectrocatalytic devices such as enzymatic biofuel cells and biosensors.

This award is funded under the American Recovery and Reinvestment Act of 2009(Public Law 111-5).

0853946
Banta

Intellectual merit

The intellectual merit of this proposal results from the first demonstration of the directed evolution of specific cell penetrating peptides (SCPPs). The directed evolution technique offers tremendous potential for the engineering of new peptides, but the critical challenge in this approach is the development of appropriate selection procedures. The PIs will use a novel bi-functional platform based on phage display technology, and this will be coupled with a physiologically relevant selection protocol. Once optimized, this platform will allow one to rapidly evolve valuable new SCPP sequences.

Broader impacts

The broader impacts of this project arise from the new experimental manipulations and therapeutic treatment options that will be made possible by the novel SCPPs. Recent research efforts have produced many exciting mechanism-based therapies. However, the delivery of these therapies to their targets has been a significant limitation. The new SCPPs will enable the expansion of the armamentarium of potential therapies for these devastating diseases. This interdisciplinary research project will also provide a fertile environment for the teaching, training, and mentoring of new engineers both at Columbia University and in the surrounding community.

1161160/ Banta

The overall goal of this innovative and high risk NSF EAGER award, funded by the Biotechnology, Biochemical and Biomass Engineering Program and by the Biosensing Program, is to use directed evolution to modify novel calcium-regulated beta roll peptides so that they can bind to protein targets with built-in allosteric recognition. The beta roll peptide has a cork-screw structure that reversibly forms in the presence of calcium. This domain has not evolved to bind to other proteins, but structurally it resembles the leucine rich repeat and ankyrin repeat proteins, which have both evolved, and been engineered, for biomolecular recognition. The researchers have begun to use directed evolution to create new beta roll peptides that bind to a model protein target, and the built-in allosteric regulation of the scaffold will enable the binding interface to be formed depending on the calcium concentration. This will be the first example of the directed evolution of an intrinsically disordered peptide for biomolecular recognition. These peptides can then be used in sensing or other molecular engineering applications.

PI's: Koder, Ronald / Banta, Scott A.
Proposal Numbers: 1403748 / 1402913
Institutions: CUNY City College / Columbia University

Title: Collaborative Research: Simplifying metabolic pathways by wiring redox proteins together

Energy transfer between enzymatic proteins is often accomplished using small molecule cofactors, such as NAD(H) and NADP(H). This system is advantageous in biological systems; however, in technology applications the use of these cofactors is undesirable for several reasons including their high cost and low stability. Advances in protein design and protein engineering have enabled tremendous advances in synthetic biology where new proteins with novel, unnatural functions can be created and characterized. In this project a new protein will be made by coupling two cofactor-dependent enzymes together. In order to eliminate the need for the cofactor, the enzymes will be connected with a designed 'staple' peptide system that contains redox center that enable electron transfer between the enzymes. This will 'wire' the proteins together so that neither one requires the unstable cofactor molecules for activity. Combined, this new enzyme complex will have an activity that is not found in nature. And this will chart a path forward for wiring other redox proteins together towards a goal of eliminating cofactor requirements in future synthetic biology projects. The team consists of PIs from City College of New York and Columbia University. There will be several outreach activities associated with this project including interactions with local high school teachers and students.

The overall goal of this collaborative project is to use protein engineering to wire two unrelated redox proteins together creating a novel cofactor-less enzymatic reaction. The investigators will attempt to demonstrate the first example of the wiring of two redox proteins together to form a ping pong redox enzyme with an activity not found in nature. They will start with two known redox enzymes: formate dehydrogenase and lactate dehydrogenase. These enzymes will be connected by a novel 'collagen staple' system that will enable specific self-assembly and the introduction of redox centers for direct electron transfer between the two enzyme active sites. The final enzyme will have a completely novel function: pyruvate reduction to lactate with the concomitant oxidation of formate to carbon dioxide. The new kinetic mechanism and activity of the resultant biocatalyst will be extensively characterized. This approach can then be extended to combine additional enzymes, creating new cofactor-less biocatalysts for industrially important oxidation and reduction reactions. This approach will allow to combine redox enzymes with sequential kinetic mechanisms into a single ping pong enzyme with a synthetic redox center. The research is integrated with educational activities which introduces undergraduates, graduate students and New York City Public High School teachers to the interdisciplinary science of biophysics. These efforts will encourage young students to pursue careers in Science, Technology, Engineering and Mathematics.

This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.

Banta, Scott
1402656
Columbia University

The separation of important molecules from complex solutions is often accomplished using proteins or peptides that have been engineered to bind the target with high affinity and selectivity. One challenge in this approach is the recovery of the target, and reuse of the binding peptide. The PI has been working with a unique peptide (called the beta roll) that is unstructured in the absence of calcium, and folds into a flattened corkscrew shape in the presence of calcium. The PI has previously engineered one face of the corkscrew for self-assembly, and has preliminary data showing the peptide can be engineered to bind to a model target protein (lysozyme). The goal of this NSF project is to develop a high throughput method to engineer new beta roll mutants that can bind to different protein targets that are important in biotechnology. The incorporation of these new engineered peptides into a bioseparations platform would be very beneficial, as it would allow for target proteins to be bound in the presence of calcium and then released upon calcium removal. This process could improve performance and reduce the costs associated with critical protein molecules, especially therapeutic proteins.

One of the key challenges in affinity-based separations is the elution of the target molecule from the affinity binding reagent. The PI proposes to expand the directed evolution approach to substantially increase the affinity of the beta roll peptides to desired targets and expand the number of targets for molecular recognition.

A method for selection from a randomized library has been developed, but higher affinity binders will require a directed evolution approach where genetic diversity is incorporated into the library. New beta roll peptides with high affinity for GFP and two common protein expression and purification tags will be researched: the maltose binding proteins (MBP) and the glutathione S transferase protein (GST). By immobilizing these evolved, high-affinity beta roll peptides on a suitable support, the PI may be able to demonstrate the use of these peptides to affinity purify MBP- and GST-tagged proteins, and use calcium chelation to elute the purified proteins. The resin can be regenerated via calcium addition and the performance of this system can be compared to traditional methods for purification with these fusion tags (amylose resin and GSH resin).

The Intellectual Merit of this proposal results from the use of a unique peptide with an intrinsic triggered conformational change as a starting scaffold for the engineering of biomolecular recognition. The calcium-induced conformational change of the beta roll peptides is a powerful molecular switch that can be exploited to reversibly disrupt engineered biomolecular interactions. Using this peptide as a starting scaffold, we will be able to generate a collection of peptides that can bind target proteins in a calcium-dependent fashion, and these new peptides will be valuable biomolecular recognition elements for affinity bioseparations.

The Broader Impact of this proposal arises from the use of protein engineering to develop a new binding motif for use in applications such as biosensors and bioseparations. The use of intrinsically disordered scaffolds for biomolecular recognition has not yet been reported in the literature, and this proposal will demonstrate that these systems can be engineered to be high affinity binders, which will be boon to those working in areas such as biosensors, smart drug delivery, bionanotechnology, and bioseparations. Funding will also be used for the mentoring of students and to continue existing outreach activities in the local community.

Principal Investigator: Scott Banta
Number: 1438263
Title: SusChEM: Long chain hydrocarbons from CO2 and electricity via genetic modification of a chemolithoautotrophic bacterium
Institution: Columbia University

There is a national need to develop carbon-neutral processes for the production of liquid transportation fuels. This project will develop a new process to turn carbon dioxide, a greenhouse gas, and renewable electricity into liquid fuels and chemicals using a unique microorganism integrated into an electrochemical device. Particular efforts will be made to dramatically increase the production of the liquid hydrocarbon heptadecane by genetically engineering the metabolism of the microorganism. Genetic engineering tools will also be developed to make the genetic modifications permanent, which will create robust cell lines capable of making fuels and chemicals in an industrial process using electricity and atmospheric carbon dioxide as the feedstock.

The goal of this project is to develop a new electrofuels platform for producing hydrocarbons from atmospheric carbon dioxide using electricity. The unique electrofuels platform consists of two integrated reactors. The first is a bioreactor containing Acidithiobacillus ferrooxidans cells which are able to grow at low pH using carbon dioxide as their sole carbon source and the oxidation of ferrous to ferric iron as an energy source. The ferric iron is sent to an electrochemical reactor which efficiently reduces the ferric iron back to ferrous iron. The combined reactor system produces biomass from electricity, water and air. The cells have been genetically modified with two different exogenous metabolic pathways for chemical and/or fuel production, and preliminary results show that cells can be transiently transformed to produce small amounts of heptadecane from carbon dioxide. In order to advance this new technology, it will be critical to increase the production of heptadecane and/or other long chain hydrocarbons. This will be accomplished using co-transformations to transiently over-express or knock-down the expression of key metabolic genes to determine strategies to direct the flux of carbon into the production of the fuels. Further development of this technology will require permanent transformation of exogenous genes into the chromosome of these cells. New methods will be developed to edit the A. ferrooxidans genome to enable the chromosomal integration of genes and pathways into this unique host cell system. The best new cell lines will be characterized in the integrated electrofuels platform so that the improvements in efficiency obtained from these maneuvers can be quantified in terms of yield of fuel per kW-hr of electricity utilized. Through these research activities, the project will train a Columbia University graduate student, who will also help to develop content for a distant-learning program to build the capacity of West African experts in the field of sustainable energy systems and solutions. Topics from this course will also be integrated into two graduate level elective courses at Columbia University as well as other outreach activities in the local community.

A few key molecules act as central electron and energy carriers inside cells. Nicotinamide adenine dinucleotide (NADH) transfers electrons and adenosine triphosphate (ATP) supplies energy. The most common method to produce ATP requires a pH gradient to be established across a membrane. The resulting flow of protons through an embedded protein regenerates ATP. This project will test the hypothesis that alternative pathways could be developed that can regenerate ATP from NADH oxidation without the need for a membrane, proton gradient, or embedded enzyme. This will require combining native and engineered enzymes to create novel cyclic pathways. These pathways will help uncover design rules required to replicate a central biological process. This technology will be critical to the development of synthetic cells. In addition, the project will involve mentoring of students and expanding existing outreach activities in the local community.

This project will design, build and test novel synthetic metabolic pathways that could regenerate ATP using energy obtained from fuel oxidation. Fuel oxidation pathways can generate reducing equivalents in the form of NADH. However other critical operations, especially those involving motion, are powered by the hydrolysis of ATP. Oxidative phosphorylation in mitochondria supplies the majority of cellular ATP from NADH oxidation and this requires the establishment of a proton gradient. Although this supplies flexibility for cellular energy processing, replicating this system presents a difficult engineering challenge. We hypothesize that oxidative phosphorylation could be replaced with novel pathways composed of kinase enzymes. These pathways will use fuel oxidation reactions to drive NADH regeneration and NAD(H) phosphorylation will drive ATP regeneration. As a proof-of-concept, an ATP-dependent reaction (firefly luciferase) powered by methanol oxidation in simple liposomes will be demonstrated. This will represent a novel energy transduction alternative to mitochondrial oxidative phosphorylation. This pathway could be easily adopted to use a wide range of potential fuels, supporting many potential future synthetic cell and biology applications.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.