Properly tuning the expression of the different polypeptide chains composing a protein complex can maximize production yields. A codon bias is observed in all organisms and contributes to efficient translation and expression of functional proteins. We applied codon de-optimization in a single multi-cistronic plasmid to improve assembly and expression of bispecific antibodies using different mammalian cell lines (Expi293, HEK, ExpiCHO and CHO cells). We also evaluated the impact of gene position on expression levels. This approach can be applied to different multispecific antibody formats and other protein complexes.

Heterologously expressed genes require adaptation to the host organism to ensure sufficient protein synthesis rates. An alignment of codon choices with the host organism’s preferred codons often results in satisfactory outcomes. To improve the method – not only for suboptimal outcomes – we study codon-specific dynamics of mRNA translation by modelling and simulation. We integrate this with machine learning approaches to propose an advanced codon optimization scheme (https://doi.org/10.1038/s41598-019-43857-5).

Recombinant soluble trimeric influenza A virus hemagglutinins (HA) and tetrameric neuraminidases (NAs) have proven to be excellent tools to decipher biological properties. Expression of HA and NA can be achieved in a plethora of different laboratory hosts. Here we report that using codon-optimized genes and sfGFP fusions, the expression yield of HA can be significantly improved. In this study, a suite of different hemagglutinin and neuraminidase constructs are described.

The yeast, Pichia pastoris, is one of the most commonly applied expression systems for heterologous protein production, even surpassing Saccharomyces cerevisiae. Here, recent synthetic biology approaches for increasing production yields will be covered ranging from artificial promoters (doi:10.1038/s41467-018-05915-w) to CRISPR-Cas systems (doi:10.1002/jcb.26474). Complementarily, also leveraging ‘classical’ comparative biology will be illustrated with the identification of novel, powerful heterologous promoters surpassing endogenous promoters in terms of both strength and regulation (doi:10.1186/s13568-020-00972-1).

Biotech scientists often get mixed results when using fusion proteins to attempt high yield production of difficult-to-express proteins. After our own experience struggling to improve yield with traditional fusion tags, we developed a new kind of fusion tag – synthetic, custom, and optimized for the protein of interest. Using this novel tag, we observed an increase in expression yields of notoriously difficult-to-express targets like amyloid beta and a designed membrane pore peptide, 2 and 10 fold, respectively when compared to SUMO.

With CHO being a good production cell line for IgG molecules, they might fail to produce more challenging candidates. The GlycoExpress (GEX®) system represents an alternative for the production difficult to express protein molecules and we will provide case studies which demonstrate the superiority in productivity and product quality.

We have developed a new method for E. coli cell extract-based, cell-free protein synthesis system. By supplementing cold shock proteins, the protein synthesis below room temperature was dramatically improved to be comparable to those at 30-37 ºC, thus it is particularly useful for expressing “difficult-to-express” proteins, which are usually prone to aggregation above room temperature. The successful applications to improving the soluble yield of aggregation-prone proteins will be presented.

Cell-free protein synthesis is a powerful technology which offers capabilities for bioproduction, biosensing, and biocomputation, freed from the constraints of living cells. In my talk, I will present recent work on the use of microfluidic devices to study and control aspects of cell-free gene expression, including synthetic transcription-factor engineering for the regulation of cell-free gene circuits, and steady-state, long-term gene expression using microfluidic chemostats.

We have developed a platform that enables multiplex investigation of tyrosine kinases and G-protein–coupled receptors (GPCRs) in cells. This is accomplished using co-translational cell-free expression of the receptor and a supporting nanolipoprotein nanodisc (NLPs).  The supporting NLP provides a platform that supports functional analysis and delivery of both GPCRs as well as tyrosine kinases directly into mammalian cells. The talk will focus on the cell-free assembly of full-length, wild-type β2AR and ERBB2 membrane bound proteins associated with NLPs in a single step process. The nano-assemblies both show protein confirmational and ligand-induced activation via. single-molecule techniques. The receptors can then be delivered onto mammalian cell membranes to affect cellular phenotypes. This is new application of NLPs that will be of interest to the scientific community.

Abcam has developed a high-throughput protein production platform that consistently delivers high-quality proteins with high success. Our first Premium Bioactive Proteins are a range of cytokines aimed at cell-culture. This session reviews how Abcam used this platform to deliver Premium Bioactive Proteins with high-quality and reliable batch to batch consistency

Biologics manufacture is expensive and time-consuming. Progression of the best candidates is key to minimising cost and time to market, however tools to assess and select the best candidates to progress remain undeveloped. This talk describes our experience generating data to produce a selection tool, through the expression, purification and analysis of a diverse set of 50 mAb sequences, and how sequence and process interact to determine titre and quality.

Targeted gene integration of protein-coding genes in the genome has become an attractive method to develop cell lines for production of therapeutic proteins. This talk will focus on the research efforts of our team to use CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE) as genome editing tools for targeted integration, and how it significantly speeds up the traditional timelines to achieve high-producing, stable CHO cell lines with predictable culture performance.