The Protein Expression Laboratory at Frederick National Laboratory for Cancer Research supports research for various National Institutes of Health entities through plasmid DNA construction, cell line development and the generation of protein reagents. Each investigator has different requirements for the final product. Depending on our clients’ aims, we plan a routing scheme for each recombinant protein target to achieve the best outcome based on previous experiments and current literature. Our decision tree relies on a vast library of expression vectors, several host expression systems, and a suite of quality control tests.

Cell-free protein synthesis decouples cell growth from protein production, enabling high-throughput production of mg quantities of protein overnight. This open, controllable system supports site-specific incorporation of multiple non-natural amino acids and efficient synthesis of complex molecules from diverse scaffolds. Scalable from bench to commercial scale, cell-free platforms accelerate high-throughput protein expression, offer precision conjugation strategies, and provide a powerful, flexible alternative to traditional cell-based biologics discovery and manufacturing.

The Trichoplusia and Spodoptera expression systems are used widely across the industry and are often only compared in terms of yield. This talk will show that are various other differences that can and should be taken into consideration when expressing a protein of interest.

Although plant biotechnology has been deployed commercially for decades for improved agronomic traits of crops, the combination of new plant expression technologies and synthetic biology components, inexpensive DNA synthesis, and novel bioprocessing strategies are enabling plants and plant cells to be used as molecular foundries.  Production platforms based on transient expression in plants within contained manufacturing facilities are showing enormous promise for rapid, scalable, and lower cost production of vaccines and therapeutics. This presentation will describe our group’s research to increase productivity and product quality in these systems while minimizing resources and production costs.

Recombinant protein production is essential for studying bacterial protein function, but many targets remain difficult to express in Escherichia coli. This presentation introduces a Rhodococcus-based expression system that enables production of challenging bacterial proteins. I will discuss practical protocols for cloning, transformation, protein expression, and purification, along with optimization and troubleshooting strategies, demonstrating how this alternative host expands the toolkit for structural and functional studies.

Robust production of biologics and cell-derived nanoparticles is vital for disease management and critical for the national security of the US and its allies. However, bioprocess complexity, costs, and instrument requirements increase when production of target bioproducts is tied to cell replication. These issues diminish reproducible biomanufacturing and production yields due to the inherent trade-off between maintaining cellular fitness and achieving robust bioproduction. Here, we engineer Cyborg Mammalian Cells, using intracellular hydrogelation, to generate semi-living micromachines that do not proliferate, but sustain biomolecule production beyond the capabilities of natural cells. We use intracellular hydrogelation, to create Cyborg Cells by forming an intracellular polymer network, restricting replication but creating a cytocompatible environment that allows the production and secretion of nanoparticles and biologics. We demonstrate the utility of Cyborg Cells as a biomanufacturing platform by showing superior biosynthesis of extracellular vesicles (EVs), viral particles, and antibodies. Our results show that Cyborg Cells produce lentivirus, EVs, and antibodies over extended periods of time using heat shock to enhance the production of functional biomolecules. Our work introduces a new biomanufacturing strategy that surpasses the limitations of traditional cell culture systems by decoupling cell replication and survival from bioproduction.