WEN GROUP
PROTEIN-ASSEMBLY ENGINEERING LAB
In contrast to a small molecule or a single protein, protein assemblies, consisting of an array of complexed or non-complexed proteins, can act in an orchestrated and synergistic manner to carry out a specific and more sophisticated biological function, such as immune-modulation and cascade catalysis. Our laboratory applies and develops interdisciplinary approaches to engineer the function of protein assemblies to solve problems in human health and sustainable energy. In the process, meaningful biological information is extracted to elucidate mechanisms underlying the problems. Ongoing projects aim to address critical issues in four distinct yet interrelated research areas.
Plant-derived biomass, especially in the forms of waste generated from various agricultural and industrial sectors, represents the most abundant, renewable, sustainable, and cost-effective raw material for production of biofuels, chemicals, and bioplastics. However, there are currently no economically feasible biorefinery in operation using waste biomass as the feedstock mainly due to two challenges. First, the waste biomass has a recalcitrant structure that locks in the sugar molecules; and second, a significant portion of the sugars is difficult to ferment into products of interest. To address both challenges, we are developing a platform technology based on multi-enzyme complexes. Distinct from most other existing methods that rely on the use of free enzymes, we assemble the enzymes into an ordered structure that enables the synergistic action of multiple enzymes with complimentary activities to achieve high biomass hydrolysis and conversion efficiency. If successful, the resulting enzyme assemblies and engineered microbial strains could enable waste-to-value conversion and reduce the production cost of ethanol by 10-20 cents per gallon.
Virus-like particles (VLPs) are supramolecular protein complexes formed by self-assembly of viral structural (envelope and/or capsid) proteins. Being “virus-like” in their 3D structure but lacking the genetic material of the virus from which they are derived, VLPs are safe and effective in inducing both humoral (antibody) and cellular (T-cell) responses. In the last decade, two prophylactic VLP vaccines have been approved for human hepatitis B virus (HBV) and papilloma virus (HPV). Unlike the success with HBV- and HPV-like particles, both of which are structurally simple and consist of only one single protein component, the production of more complex VLPs is challenging. In addition, there is no high-throughput system available to engineer the function of VLPs. Currently, we are developing a low-cost, high-throughput engineering system for producing and screening influenza-like particles that can elicit broad protective immune responses against many flu strains. In addition, we are engineering the properties of rotavirus-like particles to improve the efficacy of oral drug delivery in children with environmental enteric dysfunction.
Cancer is responsible for ~25% of all deaths in the US, mainly due to a high relapse rate, thus the idea of using a patient’s own tumor-specific T cells to keep the malignancy in check is very compelling. While this approach holds great promise, as recognized by the recent FDA approval of such a cellular cancer immunotherapy in 2010, the therapeutic efficacy is limited in part by the sub-optimal T-cell activation in vivo or ex vivo. T-cell activation and its functional outcome depend on not only the recognition of the (tumor) antigen, but also the context in which the antigen is recognized. This context, termed immunological synapse (IS), at the APC-T-cell interface plus the soluble cytokine signals determines the specificity, activation, and function of the T cell. A productive IS formation involves an array of receptor-ligand interactions that dynamically organize into a supramolecular structure. Aiming at better functional conditioning of tumor-specific T cells, we are developing protein nano-machines that allow the precise control and presentation of supramolecular patterns mimicking those found in the natural immunological synapse. The modular nature of these nano-machines also provides us a valuable tool to understand, optimize, and achieve better functional conditioning of tumor-specific, and theoretically any antigen-specific, T cells.
T cells are important immune cells for fighting infection and cancer, and as such, monitoring their behavior represents a critical step in understanding and engineering T-cell responses. T cells detect pathogens with exquisite specificity by recognizing short peptide fragments (epitopes) of pathogen-derived proteins, complexed with MHC molecules. It is estimated that there are ~107 T-cell specificities in a human body to ensure good epitope coverage for pathogen recognition. Upon antigen recognition and stimulation, T cells secrete an array of cytokines that orchestrate the action of other white blood cells for infection clearance, or directly inducing apoptosis of infected cells. Circulating T cells exhibit a range of phenotypic and functional states based on their relative level of antigen experience. As a result of this highly heterogeneous nature, it is challenging to study T cells on a systems level, especially when the availability of patient samples is usually limited. We have successfully applied state-of-the-art mass cytometry (named CyTOF, specifically Helios) and heavy metal labeled antibodies to simultaneously measure 40 parameters on single human T cells. To handle the resulting large dataset, we developed statistical and visualization algorithms to effectively distill meaningful biological information, revealing “patterns” and “fingerprints” associated with cytomegalovirus seropositivity. Currently, we are exploring strategies to further expand the detection capacity to a few hundred or more.
The Hyperion imaging mass cytometer has the capability to detect ~40 protein markers simultaneously, allowing for deep interrogation of tissues, tumors, and cell spreads at subcellular resolution. With this technology, we are able to probe complex cellular phenotypes and their relationships with the tissue microenvironment that will provide us new insights into the mechanisms of cancers, organ transplant rejection, and immune-mediated diseases.
Helios is a time-of-flight mass cytometer (CyTOF) that can measure more than 40 markers per cell. The cellular targets are labeled with metal-tagged antibodies, detected and quantified by CyTOF mass spectrometry. The high purity and choice of metal isotopes provide minimal background noise from signal overlap or endogenous cellular components. We are using Helios to do deep profiling and single-cell proteomics studies in search of molecular and cellular signatures of various diseases including cancers, autoimmunity and infectious diseases.
We use the flexible and intuitive ÄKTA Pure chromatography system for fast purification of proteins, peptides, and nucleic acids from microgram to gram levels of target product. We also find extensive use of this equipment in the purification of the VLPs synthesized in the lab.
The Attune NxT Flow Cytometer is a benchtop cytometer that uses acoustic pressure to confine the injected particles to a tight central line along the axis of the capillary as the sample passes through the optical cell for interrogation.
The Branson homogenizer is a versatile laboratory unit suitable for a broad range of liquid processing applications such as biological cell disruption / homogenization, emulsification, reaction acceleration, dispersion, fine mixing and degassing. We use this instrument extensively for cell lysis for the release of intracellularly expressed products.
Simple and efficient, the Beckman Coulter XL-100K Ultracentrifuge is designed to perform up to 100,000 rpm at up to 802,000g with a 100Ti rotor. The Wen Group performs high-purity subcellular and virus/VLP separations using this equipment.
The Wen Group uses the biosafety cabinet with 99.99% HEPA filtration for mammalian cell culture, insect cell culture and viral stock manipulation for the purpose of personnel, environment and product protection.
The Wen Group frequently runs polymerase chain reactions (PCR) for the purpose of cloning and so the Bio-Rad thermal cycler is an essential component of our lab. This instrument employs the Peltier effect technology for precise temperature control and thermal gradient to easily optimize PCR assays in a single run.
The electrophoresis bay includes horizontal and vertical electrophoresis tanks, tunable BioRad power packs, magnetic stirrer and stock running buffers, all used to run a large number of key characterization assays such as DNA gel quantification, SDS-PAGE and Western Blotting.
With a maximum speed of 29,000 RPM and a total capacity of 6x1000mL, the Sorvall centrifuge is used to perform simple high-speed separations, such as pelleting transformed cells cultured in suspension media on a large scale (on the order of liters).
The NanoDrop is a microvolume UV/Vis spectrophotometer designed for the analysis of DNA, RNA and proteins. It measures 1 μL samples with high accuracy and reproducibility, utilizing a unique patented sample retention technology that employs only surface tension to hold the sample in place, eliminating any need for cuvettes or large sample volumes. It also has cuvette capability. Working extensively with protein engineering and assembly on a small scale, the Wen Group finds significant use of this instrument for protein quantification.
The Agilent UV/Vis Spectrophotometer features rapid scan and measurement rates which are ideal for kinetic studies. It can be equipped with an optical fiber pick-up/return accessory. The Wen Group primarily uses this instrument to perform enzyme activity/kinetics studies.
The Gel Doc EZ system is a compact and automated gel imaging instrument designed to yield publication-quality images and analyzed results. The Wen Group uses this instrument with the UV tray for ethidium bromide staining of DNA gels and fluorescence imaging of protein gels using SYPRO orange, and the white tray for imaging Coomassie blue stained protein gels.
The I26 rotary shaker shown here is an incubated/refrigerated shaker equipment used extensively by the Wen Group for maintenance of desired thermal conditions for the large scale suspension culture of transformed yeast cells. We also have two Excella 24 rotary shakers (not pictured) used for the culture of insect cells and prokaryotic cells respectively.
Among other applications, we use the Eppendorf 5810 R large centrifuge to spin down microplates, large volume tissue samples and for protein concentration. The refrigerated microcentrifuge is used to spin down temperature-sensitive specimens such as proteins, peptides, DNA, cell samples, etc.
Other equipments include an inverted microscope, a liquid nitrogen storage tank, -80°C freezer, -20°C freezer, cell culture 4°C fridge, water baths (32°C, 37°C and 42°C) and chemical fume hoods.
