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.

Lactococcus lactis, a GRAS bacterium, emerged in the last years as an good alternative for soluble (therapeutic proteins and antigens) and membrane protein expression and characterizations. It’s an interesting host because of its moderate proteolytic activity, absence of inclusion-body and endotoxin, and MP targeting into a single plasma membrane. Using NICE system, almost 100 MPs were successfully expressed. Finally, L. lactis represents a promising and efficient system for protein expression.

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.

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.

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.

Potency assays are critical for demonstrating the mechanism of action of therapeutic drugs and biologics throughout clinical development. To ensure a reliable supply of critical potency assay reagents for an immunology biotherapeutic program, we developed stable CHO cell lines for in house production of ligand and receptor. Both proteins were generated by transient transfection using the original control process, serving as an orthogonal benchmark for comparison. Comprehensive analytical characterization confirmed high comparability to existing control materials. This strategy reduces lot to lot variability, enhanced robustness and reproducibility, and establishes a scalable, sustainable approach to reagent lifecycle management throughout clinical development.

Ribosomal RNA (rRNA) heterogeneity shapes translation efficiency, and could be altered to tune protein expression. We constructed an isogenic rRNA E. coli strain by reverting 134 polymorphisms across six operons. Despite prior in vitro data showing substantial translational differences from these rRNA micro-heterogeneities, isogenic strains matched wild-type fitness and protein yields in vivo and in cell-free systems. This chassis enables future ribosome engineering efforts toward enhanced therapeutic biomanufacturing.

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.

Optogenetics offers unprecedented opportunities to dynamically control microbial production with light for improved process performance. This presentation will highlight recent advances demonstrating methanol-free, light-controlled recombinant protein production in Komagataella phaffii and the development of photoswitchable protein binders for light-triggered affinity purification. Together, these technologies illustrate how optogenetics can transform both upstream bioprocessing and downstream purification, enabling more precise, sustainable, and integrated production workflows.