A concise guide to choosing suitable gene expression systems for recombinant protein production.

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making.

Inducible protein expression in piggyBac transposase mediated stable HEK293 cell pools.

Described here is the use of piggyBac transposase generated HEK293 stable cell pools for doxycycline-inducible protein production. The key benefits of the system are that low amounts of plasmid DNA are needed for transfection, high levels of protein expression can be achieved also for toxic proteins at robust scalability and reproducibility and the recombinant cell line can be stored as frozen cell bank. Transfection, selection, expression and purification of enhanced green fluorescence protein (eGFP) and SARS-CoV-2 Spike protein are described in this chapter.

A fast-track protocol for protein expression using the BEV system.

Baculovirus-insect cell expression (BEV) has become one of the most widely used eukaryotic systems for heterologous protein expression. The combination of engineered baculovirus genomes together with a variety of compatible vectors, robust insect cell lines, serum-free media and commercial kits have made it a standard workhorse in many "non-virology-expert" laboratories. Despite these significant improvements, the BEV system still has major drawbacks, primarily the time required to amplify recombinant virus and its inherent instability.

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method.

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins.

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function.

Baculovirus-driven protein expression in insect cells: A benchmarking study.

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production.

A new single-step protocol for rapid baculovirus-driven protein production in insect cells.

Background: In the last three decades, the Baculovirus expression vector system (BEV) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. Despite the significant improvements introduced during the past years, the BEV system still has major drawbacks, primarily the time required to generate recombinant virus and virus instability for certain target proteins. In this study we show that the conventional method to generate recombinant Baculovirus using a Tn7 transposition based system can be shortened to a single-step transfection-only procedure without further amplification.

A new method to customize protein expression vectors for fast, efficient and background free parallel cloning.

Background: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.

Identification and functional validation of novel autoantigens in equine uveitis.

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading.

Differential modification of phosducin protein in degenerating rd1 retina is associated with constitutively active Ca2+/calmodulin kinase II in rod outer segments.

Retinitis pigmentosa comprises a heterogeneous group of incurable progressive blinding diseases with unknown pathogenic mechanisms. The retinal degeneration 1 (rd1) mouse is a retinitis pigmentosa model that carries a mutation in a rod photoreceptor-specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retinae is crucial for explaining this degeneration and could assist in suggesting novel therapies.

Proteomic profiling of primary retinal Müller glia cells reveals a shift in expression patterns upon adaptation to in vitro conditions.

Cultured primary retinal Müller glia cells (RMG), a glia cell spanning the entire neuroretina, have recently gained increased attention, especially with respect to their presumed in vivo role in supporting photoreceptor function and survival. Cultured RMG cells, however, are at risk to lose much of their in vivo features. To determine the conditions of isolated primary RMG cells best corresponding with their physiological role in the intact retina, we profiled the respective proteomes of RMG freshly isolated from intact pig eye, as well as from cultured material at different timepoints.

Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells.

Purpose: Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells.