Escherichia coli, Saccharomyces cerevisiae, and mammalian culture cells are standard host organisms for genetic engineering and biological research. We developed a yeast expression plasmid that enables expression of the cloned gene in E.coli and mammalian cells via the transfer of PCR products amplified from the plasmid as a template. The concept is PCR-mediated gene expression using end-promoter constructs. This is the first all-in-one plasmid applicable for expressions in three host organisms.

Single nucleotide variants (SNVs) are the underpinnings for human genomic diversity. While nonsynonymous variants are appreciated for transforming the protein sequence, synonymous variants have only lately gained increased recognition for altering mRNA structure, splicing, miRNA binding, co-translational protein folding, and protein stability. In my talk, I will show examples of single or multiple synonymous mutations in the form of codon optimization or codon recoding that can change protein attributes.

The designing of microbial genomes remains challenging due to the complexity of biology. Adaptive Laboratory Evolution (ALE) leverages nature’s problem-solving processes to generate optimized genotypes currently inaccessible to rational methods. This study describes how novel strain designs can be extracted from aggregated ALE data by designing, building, and testing novel Escherichia coli strains. These results demonstrate how strain design efforts can be enhanced by the meta-analysis of aggregated ALE data.

Bacterial lysate-based cell-free systems are promising platforms for protein expression. Currently the total expression capacity of these systems can be quite limited. Here, we explore the residual metabolic network in cell-free lysates and identify the impacts of this "endogenous" metabolism on system properties including rate of protein production and final expression titers. We used metabolomics to characterize metabolic dynamics and identified that the main driver of chemical changes in cell-free lysates is endogenous metabolism, not protein expression, and that endogenous metabolism can have a strong negative effect on total protein production.

The overpopulation of the planet has caused a massive need for energy and reduced carbon, and microalgae, which are the most efficient producers within the ecosystem, can be utilized to satisfy mankind’s demands. Algae biotechnology has generated varied products such as therapeutics, nutraceuticals, food, polymers, and biofuels. But to provide commercially viable bioproducts we need to advance two principal technologies: powerful genetic tools, and enhanced microalgal cultivation techniques. We utilize synthetic biology to generate strong, inducible promoters that increase the yields of recombinant products, and bioreactors or open pond cultivation techniques depending on the product of interest.                   

Production of recombinant proteins in E. coli is the engine of many drug discovery efforts and is often the rate-limiting step. Another bacterial host Vibrio natriegens has recently shown promise, as an alternative to E. coli. Here, we present a set of general protocols for its use. Moreover, we show that Vibrio natriegens can outproduce E. coli for certain protein reagents, and or solubilize protein where E. coli fails.

The Institute for Protein Design has developed powerful processes for computational protein design, most recently the Diffusion model, which combines structural prediction networks with generative diffusion with the ability to generate highly accurate designs optimized for soluble expression. This results in a high numbers of  proteins requiring experimental validation.  The IPD has developed methods to express, purify, and characterize these designed proteins that can keep pace with design velocity. These methods include utilization of open source robotics, intentional scaling of culture volumes and fractionation for streamlined operations, and high throughput analytical methods for the evaluation of protein aggregation and oligomerization.

The Just-Evotec Biologics CLD platform is optimized to decrease development timelines and increase throughput by using automation from transfection through RCB creation. Our high-throughput transfection method allows us to simultaneously screen 96 transfectants in stable pools to identify more manufacturable molecules with a reduced timeline. Using automation, we are capable of screening over 350 clones allowing us to identify cell lines with high productivity and favorable product quality attributes.

The expression screening of large numbers of protein constructs can be automated utilizing the baculovirus expression system (BEVS) and TECAN automation. Biophysical characterization of small-scale screening samples, such as aSEC and nanoDSF,  provides a more robust screening funnel with better prediction of successful clones than simple SDS-page analysis. Additionally, expanding to an automated “midi-scale” screen allows for production of sufficient material to perform more in-depth POC studies such as Biacore, MST, and LC-MS.

Epigenetics is an important emerging field in biotechnology. The authors examine DNA methylation and transcription profiles of two different CHO hosts and the resultant production clones. Combining transcriptomics with DNA methylation data enables identification of potential processes and factors that may contribute to the differences in cell physiology between different production hosts. These differences, including epigenetic writers, readers, erasers, and effectors in turn may be important to explaining the variability in productivities of these cell lines.

Glycoprotein-based drugs represent the fastest-growing class of therapeutic molecules. As a key critical quality attribute of recombinant proteins, glycosylation plays a significant role in maintaining the stability, safety and efficacy of such molecules. Utilizing a platform that combines glycoengineering and systems biology, we seek to develop recombinant products that contain optimal glycan compositions and demonstrate improved biophysical characteristics and biological activity.

Expressing complex molecules such as bispecific antibodies and fusion proteins often faces challenges like low titer, poor product quality, and difficult-to-control post-translational modifications. We have tackled these challenges by engineering the expression host and vectors and optimizing the culture conditions. 

CHO cell lines have significant phenotypic variability though derived from a single cell progenitor. This variability may lead to or be indicative of the propensity of a cell line to exhibit production instability over time. Here we characterize RNA expression from stable and unstable cell lines using single-cell RNA sequencing. Our work shows that clonally derived cell lines are a complex metapopulation of cells whose make-up changes significantly with age.