Construct design towards soluble protein fragments for biochemical, biophysical, and structural analyses has been greatly facilitated by algorithms predicting features such as domains, disorder, and secondary elements. The recent advent of AI tools such as AlphaFold2, however, has transformed in silico structural biology. Here we present practical tips for using bioinformatics and AI tools in construct design to help users improve the likelihood of obtaining functional proteins for their needs.

Expanding toolbox options to produce multispecifics to increase protein yields and promote correct molecule formation during expression can alleviate downstream purification bottlenecks. Targeted Dual Selection (TDS) with transposon guided semi-targeted gene integration can be used to optimize stably-expressed pools to enhance protein titers and pairing immediately off capture purification. We demonstrate that transient data can effectively predict vector combinations for larger scale TDS stable pools. Utilizing this technology early in research can simplify purification strategies and increase production yields, facilitating successful project progressions. 

Bacterial hosts like E. coli enable rapid, cost-effective protein production but face challenges such as misfolding, aggregation, toxicity, and the need for complex modifications like disulfide bonds. Here, we introduce PandaPure, a synthetic organelle framework driven by liquid-liquid phase separation that "eukaryotizes" E. coli, transforming its capabilities for streamlined expression and purification. This approach simplifies workflows, improves expression success rates, and delivers high-purity proteins through straightforward extraction. By bridging the strengths of bacterial and eukaryotic systems, PandaPure offers a scalable, efficient, and transformative solution to longstanding challenges in protein production for research, diagnostics, and industrial applications.

The Modular Protein Expression Toolbox (MoPET) empowers protein engineers with a flexible and efficient approach to generate both single-expression constructs and combinatorial libraries. The system utilizes standardized, reusable DNA modules, enabling rapid and reliable assembly of constructs tailored to diverse experimental needs. This presentation highlights MoPET's capabilities, offering practical insights and real-world use cases to demonstrate its potential to transform protein expression workflows and accelerate discovery.

Pichia pastoris is a low-cost expression host used to produce protein-based therapeutics. Two major drawbacks of this system are low-protein secretion levels and proteolysis of the product during fermentation. We overcame these shortcomings by formulating a new chemically-defined growth medium and using site saturation mutagenesis. These changes increased the secretion of intact multivalent miniproteins, containing multiple target specificities codified within a single gene, by more than 20-fold.

Pichia pastoris is known for secreted protein production. To enhance the development workflow, a pool-based screening method was developed following strategies utilized in CHO cell line development. Expression of multiple protein and signal sequences were evaluated to identify optimal conditions for protein secretion. Individual strains from high, medium, and low-producing pools demonstrated comparable titer to their corresponding pool. This approach enabled a wide experimental design space with minimal resources.

Sutro Biopharma’s cell-free protein synthesis system is a powerful platform to produce antibodies containing non-natural amino acids (nnAAs) that facilitates homogenous site-specific conjugation of antibody-drug conjugates (ADCs). This talk will highlight the cell-line engineering approaches used to enable the GMP manufacturing of IgGs containing nnAAs for clinical ADC production, including ADCs bearing two payloads with different mechanisms of action and ADCs with high drug-to-antibody ratios (>8).

We have developed a cell-free protein production pipeline that significantly accelerates the synthesis of therapeutic proteins, including cytokines, antibodies, and membrane proteins. The automated system produces microgram-to-milligram quantities of multiple proteins simultaneously, enabling advanced characterization methods, including in-cell NMR. The presentation highlights key innovations that streamline protein production and interaction studies, offering substantial advancements for the design, development, and optimization of biotherapeutic candidates.

Lactate production is ubiquitous in proliferative mammalian cells and poses challenges for biopharmaceutical production as its accumulation inhibits cell growth and protein production. We have eliminated lactate production in CHO cells by knocking out multiple genes using CRISPR/Cas9. These cells remain amenable for the production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining glycosylation. We will also share an initial assessment of the biological impact of this knockout, including how metabolism and transcriptional profiles are perturbed.

Transient protein production in mammalian cells is vital for biopharmaceutical industry, enabling rapid molecule generation for early-stage biologics discovery. We present an optimized transfection protocol using high-density HD-CHO cell cultures enhanced by dimethylacetamide (DMA) and mild hypothermia. Our Design of Experiments (DOE) approach has doubled protein titers, streamlined the workflow, and minimized complexity. This presentation shares insights into our optimization method and its potential to accelerate protein production development.