Expression In Mammalian Cells Research Articles

Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid.

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

Circular single-stranded DNA as switchable vector for gene expression in mammalian cells

Synthetic gene networks in mammalian cells are currently limited to either protein-based transcription factors or RNA-based regulators. Here, we demonstrate a regulatory approach based on circular single-stranded DNA (Css DNA), which can be used as an efficient expression vector with switchable activity, enabling gene regulation in mammalian cells. The Css DNA is transformed into its double-stranded form via DNA replication and used as vectors encoding a variety of different proteins in a wide range of cell lines as well as in mice. The rich repository of DNA nanotechnology allows to use sort single-stranded DNA effectors to fold Css DNA into DNA nanostructures of different complexity, leading the gene expression to programmable inhibition and subsequently re-activation via toehold-mediated strand displacement. The regulatory strategy from Css DNA can thus expand the molecular toolbox for the realization of synthetic regulatory networks with potential applications in genetic diagnosis and gene therapy.

Comprehensive Investigation of Recombinant Human TMPRSS4 Expression and Purification Across Diverse Expression Platforms: Bacterial, Insect (BVES), and Mammalian Systems

TMPRSS4, an essential transmembrane protease serine 4, holds significant relevance in diverse biological contexts, making it a molecule of interest across various fields. This transmembrane serine protease, TMPRSS4, plays a pivotal role in multiple areas, including cancer research, disease processes, and potentially in the spread of viral infections. In light of its multifaceted significance, the research and production of active TMPRSS4 protein serve as a critical foundation for in-depth structural and functional studies, ultimately leading to the identification of potential inhibitors. Within this context, our study encompasses an extensive investigation of various expression systems, including microbial, insect cells, and mammalian cells, aimed at obtaining biologically active TMPRSS4 protein. This study underscores our efforts in optimizing solubility. We explored different E. coli expression strains, optimized expression conditions at various temperatures, employed both inducible and autoinduction-based approaches, fine-tuned the Multiplicity of Infection (MOI), and controlled cell density in Sf9 cells using BVES. Additionally, we delved into the exploration of alternative signal peptides for secretory expression in mammalian cells, followed by the characterization of purified protein variants. Through rigorous biochemical analyses and functional assays, we discovered the distinct advantages associated with mammalian cell expression, ultimately resulting in the production of biologically active TMPRSS4. Our findings provide critical insights into the optimization of expression systems, thereby enhancing the functionality of the protein.

SHIELD: a platform for high-throughput screening of barrier-type DNA elements in human cells

Chromatin boundary elements contribute to the partitioning of mammalian genomes into topological domains to regulate gene expression. Certain boundary elements are adopted as DNA insulators for safe and stable transgene expression in mammalian cells. These elements, however, are ill-defined and less characterized in the non-coding genome, partially due to the lack of a platform to readily evaluate boundary-associated activities of putative DNA sequences. Here we report SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains), a platform tailored for the high-throughput screening of barrier-type DNA elements in human cells. SHIELD takes advantage of the high specificity of serine integrase at heterochromatin, and exploits the natural heterochromatin spreading inside lamina-associated domains (LADs) for the discovery of potent barrier elements. We adopt SHIELD to evaluate the barrier activity of 1000 DNA elements in a high-throughput manner and identify 8 candidates with barrier activities comparable to the core region of cHS4 element in human HCT116 cells. We anticipate SHIELD could facilitate the discovery of novel barrier DNA elements from the non-coding genome in human cells.

Effect of human β-Globin second intron on transient gene expression in mammalian cell lines

Exogenous protein expression in mammalian cells is necessary to produce therapeutic proteins and modern medical applications like developing DNA vaccines and gene therapy. This study examines the human-Globin (hBG) second intron's capacity for intron-mediated enhancement (IME) in various mammalian cell lines. Our study's main aim is to investigate the effect of the incorporation and arrangement of the second intron of the human Beta-globin gene into the pVAX-1 expression cassette on improving the expression of foreign genes. Two plasmids were constructed, one with the hBG second intron positioned upstream and the other downstream in the expression cassette. EGFP expression was evaluated at the mRNA and protein levels after transfection using Lipofectamine 2000 using One-way ANOVA analysis. Results showed that the pVAX-1 harbouring the hBG second intron did not lead to enhanced transient EGFP expression and did not exhibit Intron Mediated Enhancement (IME) in tested mammalian cell lines. Further investigations are necessary to understand factors contributing to the lack of enhancement and explore alternative intron options for optimizing foreign gene expression in cell lines.

Expression, Purification, and Crystallization of the Vγ9Vδ2 T-cell Receptor Recognizing Protein/Peptide Antigens.

γδ T cells, especially Vγ9Vδ2 T cells, play an important role in mycobacterial infection. We have identified some Vγ9Vδ2 T cells that recognize protein/peptide antigens derived from mycobacteria, which may induce protective immune responses to mycobacterial infection. To clarify the structural basis of the molecular recognition mechanism, we tried many methods to express the Vγ9Vδ2 T-cell receptor (TCR). The Vγ9Vδ2 TCR was not expressed well in a prokaryotic expression system or a baculovirus expression system, even after extensive optimization. In a mammalian cell expression system, the Vγ9Vδ2 TCR was expressed in the form of a soluble heterodimer, which was suitable for crystal screening. Reduced-temperature cultivation (cold shock) increased the yield of the recombinant TCR. The recombinant purified TCR was used for crystal trials, and crystals that could be used for X-ray diffraction were obtained. Although we have not yet determined the crystal structure of the Vγ9Vδ2 TCR, we have established a procedure for Vγ9Vδ2 TCR expression and purification, which is useful for basic research and potentially for clinical application.

Colorectal cancer-associated mutations impair EphB1 kinase function

Erythropoietin-producing hepatoma (Eph) receptor tyrosine kinases regulate the migration and adhesion of cells that are required for many developmental processes and adult tissue homeostasis. In the intestinal epithelium, Eph signaling controls the positioning of cell types along the crypt-villus axis. Eph activity can suppress the progression of colorectal cancer (CRC). The most frequently mutated Eph receptor in metastatic CRC is EphB1. However, the functional effects of EphB1 mutations are mostly unknown. We expressed and purified the kinase domains of WT and five cancer-associated mutant EphB1 and developed assays to assess the functional effects of the mutations. Using purified proteins, we determined that CRC-associated mutations reduce the activity and stability of the folded structure of EphB1. By mammalian cell expression, we determined that CRC-associated mutant EphB1 receptors inhibit signal transducer and activator of transcription 3 and extracellular signal-regulated kinases 1 and 2 signaling. In contrast to the WT, the mutant EphB1 receptors are unable to suppress the migration of human CRC cells. The CRC-associated mutations also impair cell compartmentalization in an assay in which EphB1-expressing cells are cocultured with ligand (ephrin B1)-expressing cells. These results suggest that somatic mutations impair the kinase-dependent tumor suppressor function of EphB1 in CRC.

Pathogenicity analysis and splicing rescue of a classical splice site variant (c.1343+1G>T) of CNOT1 gene associated with neurodevelopmental disorders.

Mutations in the CNOT1 gene lead to an incurable rare neurological disorder mainly manifested as a clinical spectrum of intellectual disability, developmental delay, seizures, and behavioral problems. In this study, we investigated a classical splice site variant of CNOT1 (c.1343+1G>T) associated with neurodevelopmental disorders, which was a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. To link CNOT1 dysfunction with the neurodevelopmental phenotype observed in a patient, in vitro minigene assay was used to verify the effect of CNOT1 gene splice site variant c.1343+1G>T on mRNA splicing. We also explored the impact of transient transfection introducing modified U1 snRNA on correcting the splicing variant. Through minigene expression in mammalian cells, we demonstrated that the variant induced complete exon 12 skipping, which explained the patient's clinical condition and provided additional genetic diagnosis evidence for the clinical significance of the variant. Moreover, we confirmed that the aberrant splice pattern could be partially corrected by the modified U1 snRNA at the mRNA level, which provided strong evidence for the therapeutic potential of modified U1 snRNA in neutralizing the hazardous effect of incorrect splicing patterns.

Identification of Two Subsets of Subcompartment A1 Associated with High Transcriptional Activity and Frequent Loop Extrusion.

Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes.

Affordable amino acid α/β-deuteration and specific labeling for NMR signal enhancement: Evaluation on the kinase p38α

Although very effective in decreasing NMR relaxation of large proteins, homogeneous deuteration can be costly, and anyway unsuitable for recombinant production in metazoan systems. We sought to explore other deuteration schemes, which would be adapted to protein expression in mammalian cells. Here, we evaluate the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α. We report the position-specific deuteration of free amino acids by using enzyme-assisted H/D exchange, executed by the cystathionine gamma-synthase and a newly designed high-performance mutant E325A. Then, we used cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly). Our results show signal enhancements up to three in 1H-15N spectra when these α/β-deuterated amino acids are integrated. Because our approach relies on single 2Hα/β-15N-amino acid labeling, an additional three-fold increase in sensitivity is obtained by the possible use of moderate resolution SOFAST-HMQC instead of the classical HSQC or TROSY experiments. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of p38α in one hour with S/N∼10.

The role of m5C methyltransferases in cardiovascular diseases.

The global leading cause of death is cardiovascular disease (CVD). Although advances in prevention and treatment have been made, the role of RNA epigenetics in CVD is not fully understood. Studies have found that RNA modifications regulate gene expression in mammalian cells, and m5C (5-methylcytosine) is a recently discovered RNA modification that plays a role in gene regulation. As a result of these developments, there has been renewed interest in elucidating the nature and function of RNA "epitranscriptomic" modifications. Recent studies on m5C RNA methylomes, their functions, and the proteins that initiate, translate and manipulate this modification are discussed in this review. This review improves the understanding of m5C modifications and their properties, functions, and implications in cardiac pathologies, including cardiomyopathy, heart failure, and atherosclerosis.

Antisense Oligonucleotide Activation via Enzymatic Antibiotic Resistance Mechanism.

The structure and mechanism of the bacterial enzyme β-lactamase have been well-studied due to its clinical role in antibiotic resistance. β-Lactamase is known to hydrolyze the β-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate β-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by β-lactamase-mediated cleavage of a cephalosporin motif capable of silencing the expression of T-box transcription factor Ta (tbxta), also referred to as no tail a (ntla), eliciting a distinct, observable phenotype. We explore the use of β-lactamase to elicit a biological response in aquatic embryos for the first time and expand the utility of cephalosporin as a cleavable linker beyond targeting antibiotic-resistant bacteria. The addition of β-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.

Optimization of mammalian expression vector by cis-regulatory element combinations.

The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot. The receptor binding domain (RBD) sequence from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein was used to replace the eGFP sequence in the expression vector and the RBD expression was detected by qRT-PCR and western blot. The results showed that protein expression can be regulated by optimizing the combination of cis-acting elements. The vector with the CMV enhancer, EF-1α core promoter, and TEF terminator was found to express approximately threefold higher eGFP than the unmodified vector in different animal cells, as well as 2.63-fold higher recombinant RBD protein than the original vector in HEK-293T cells. Moreover, we suggest that combinations of multiple regulatory elements capable of regulating gene expression do not necessarily exhibit synergistic effects to enhance expression further. Overall, our findings provide insights into biological applications that require the regulation of gene expression and will help to optimize expression vectors for biosynthesis and other fields. Additionally, we provide valuable insights into the production of RBD proteins, which may aid in developing reagents for diagnosis and treatment during the COVID-19 pandemic.

BMP7 expression in mammalian cortical radial glial cells increases the length of the neurogenic period.

The seat of human intelligence is the human cerebral cortex, which is responsible for our exceptional cognitive abilities. Identifying principles that lead to the development of the large-sized human cerebral cortex will shed light on what makes the human brain and species so special. The remarkable increase in the number of human cortical pyramidal neurons and the size of the human cerebral cortex is mainly because human cortical radial glial cells, primary neural stem cells in the cortex, generate cortical pyramidal neurons for more than 130 days, whereas the same process takes only about 7 days in mice. The molecular mechanisms underlying this difference are largely unknown. Here, we found that bone morphogenic protein 7 (BMP7) is expressed by increasing the number of cortical radial glial cells during mammalian evolution (mouse, ferret, monkey, and human). BMP7 expression in cortical radial glial cells promotes neurogenesis, inhibits gliogenesis, and thereby increases the length of the neurogenic period, whereas Sonic Hedgehog (SHH) signaling promotes cortical gliogenesis. We demonstrate that BMP7 signaling and SHH signaling mutually inhibit each other through regulation of GLI3 repressor formation. We propose that BMP7 drives the evolutionary expansion of the mammalian cortex by increasing the length of the neurogenic period.

Akkermansia muciniphila and its membrane protein ameliorates intestinal inflammatory stress and promotes epithelial wound healing via CREBH and miR-143/145

BackgroundThe intestinal epithelial barrier is the interface for interaction between gut microbiota and host metabolic systems. Akkermansia muciniphila (A. muciniphila) is a key player in the colonic microbiota that resides in the mucus layer, whose abundance is selectively decreased in the faecal microbiota of inflammatory bowel disease (IBD) patients. This study aims to investigate the regulatory mechanism among A. muciniphila, a transcription factor cAMP-responsive element-binding protein H (CREBH), and microRNA-143/145 (miR-143/145) in intestinal inflammatory stress, gut barrier integrity and epithelial regeneration.MethodsA novel mouse model with increased colonization of A muciniphila in the intestine of CREBH knockout mice, an epithelial wound healing assay and several molecular biological techniques were applied in this study. Results were analysed using a homoscedastic 2-tailed t-test.ResultsIncreased colonization of A. muciniphila in mouse gut enhanced expression of intestinal CREBH, which was associated with the mitigation of intestinal endoplasmic reticulum (ER) stress, gut barrier leakage and blood endotoxemia induced by dextran sulfate sodium (DSS). Genetic depletion of CREBH (CREBH-KO) significantly inhibited the expression of tight junction proteins that are associated with gut barrier integrity, including Claudin5 and Claudin8, but upregulated Claudin2, a tight junction protein that enhances gut permeability, resulting in intestinal hyperpermeability and inflammation. Upregulation of CREBH by A. muciniphila further coupled with miR-143/145 promoted intestinal epithelial cell (IEC) regeneration and wound repair via insulin-like growth factor (IGF) and IGFBP5 signalling. Moreover, the gene expressing an outer membrane protein of A. muciniphila, Amuc_1100, was cloned into a mammalian cell-expression vector and successfully expressed in porcine and human IECs. Expression of Amuc_1100 in IECs could recapitulate the health beneficial effect of A. muciniphila on the gut by activating CREBH, inhibiting ER stress and enhancing the expression of genes involved in gut barrier integrity and IEC’s regeneration.ConclusionsThis study uncovers a novel mechanism that links A. muciniphila and its membrane protein with host CREBH, IGF signalling and miRNAs in mitigating intestinal inflammatory stress–gut barrier permeability and promoting intestinal wound healing. This novel finding may lend support to the development of therapeutic approaches for IBD by manipulating the interaction between host genes, gut bacteria and its bioactive components.

Optimization of genetic code expansion in the baculovirus expression vector system (BEVS)

The production of recombinant proteins containing unnatural amino acids, commonly known as genetic code expansion (GCE), represents a breakthrough in protein engineering that allows for the creation of proteins having novel designed properties. The naturally occurring orthogonal pyrrolysine tRNA/aminoacyl-tRNApyl synthetase pair (tRNApyl/PylRS) found in Methanosarcinaceae species has provided a rich platform for protein engineers to build a library of amino acid derivatives suitable for the introduction of novel chemical functionalities. While reports of the production of such recombinant proteins utilizing the tRNApyl/PylRS pair, or mutants thereof, is commonplace in Escherichia coli and mammalian cell expression systems, there has only been a single such report of GCE in the other stalwart of recombinant protein production, the baculovirus expression vector system (BEVS). However, that report formulates protein production within the designs of the MultiBac expression system [1]. The current study frames protein production within the strategies of the more commonplace Bac-to-Bac system of recombinant baculovirus production, via the development of novel baculovirus transfer vectors that harbor the tRNApyl/PylRS pair. The production of recombinant proteins harboring an unnatural amino acid(s) was examined using both an in cis and an in trans arrangement of the tRNApyl/PylRS pair relative to the target protein ORF i.e. the latter resides, respectively, on either the same vector as the tRNApyl/PylRS pair, or on a separate vector and deployed in a viral co-infection experiment. Aspects of the transfer vector designs and the viral infection conditions were investigated.

Bioactivity of recombinant humanized monoclonal antibody against HER2 in-vivo and in-vitro and its mechanism of action in ovarian cancer.

Human epidermal growth factor receptor 2 (HER2) protein is overexpressed on the surface of various epithelial ovarian cancer tissues, mediates the proliferation, differentiation, metastasis, and signal transduction of tumor cells, and thus is a potential cancer therapeutic target. However, its research in ovarian cancer is still limited, and how to quickly obtain a large number of antibodies remains a concern for researchers. In this study, we expressed the recombinant anti-HER2 humanized monoclonal antibody (rhHER2-mAb) in human embryonic kidney 293 (HEK293) cells through transient gene expression (TGE) technology by constructing a mammalian cell expression vector. Firstly, the transfection conditions has been optimized, the ratio of light chain (LC) and heavy chain (HC) was optimized in the range of 4:1 to 1:2 and the ration DNA and polyethyleneimine was optimized in the range of 4:1 to 1:1. The antibody was purified by rProtein A affinity chromatography, and its mediated antibody-dependent cellular cytotoxicity (ADCC) was identified by lactate dehydrogenase release assays. The anti-tumor activity of rhHER2-mAb was evaluated in non-obese diabetic/severe combined immunodeficiency mice. The expression of rhHER2-mAb in the HEK293F cells was at the highest level (100.5 mg/L) when the DNA/polyethyleneimine and light-chain/heavy-chain ratios were 1:4 and 1:2, respectively. The half-maximal inhibitory concentration of the ADCC of the antibodies against the SK-OV-3, OVCAR-3, and A-2780 cells were 12.36, 5.43, and 102.90 ng/mL, respectively. The animal experiments with the mice showed that rhHER2-mAb effectively inhibited the growth (P<0.01) of the SK-OV-3 tumors at a dose of 10 mg/kg. TGE technology allows us to quickly obtain a large number of anti-HER2 antibodies compared to the traditional method of constructing stable cell lines, and its in vitro and in vivo studied shows that our anti-HER2 antibody have higher affinity and better biological activity bioactivity (P<0.01) compared to Herceptin. Our findings provide novel insights into the development and production of future biotechnology-based drugs using the TGE technology of HEK293F.

Robust Protocol for the Synthesis of BSA Nanohydrogels by Inverse Nanoemulsion for Drug Delivery.

In a highly efficient and reproducible process, bovine serum albumin (BSA) nanogels are prepared from inverse nanoemulsions. The concept of independent nanoreactors of the individual droplets in the nanoemulsions allows high protein concentrations of up to 0.6% in the inverse total system. The BSA gel networks are generated by the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride coupling strategy widely used in protein chemistry. In a robust work-up protocol, the hydrophobic continuous phase of the inverse emulsion is stepwise replaced by water without compromising the colloidal stability and non-toxicity of the nanogel particles. Further, the simple process allows the loading of the nanogels with various cargos like a dye (Dy-495), a drug (ibuprofen), another protein [FMN-binding fluorescent protein (EcFbFP)], and oligonucleotides [plasmid DNA for enhanced GFP expression in mammalian cells (pEGFP c3) and a synthetic anti-Pseudomonas aeruginosa aptamer library]. These charged nanoobjects work efficiently as carriers for staining and transfection of cells. This is exemplarily shown for a phalloidin dye and a plasmid DNA as cargo with adenocarcinomic human alveolar basal epithelial cells (A549), a cell revertant of the SV-40 cancer rat cell line SV-52 (Rev2), and human breast carcinoma cells (MDA-MB-231), respectively.

CysPresso: a classification model utilizing deep learning protein representations to predict recombinant expression of cysteine-dense peptides

BackgroundCysteine-dense peptides (CDPs) are an attractive pharmaceutical scaffold that display extreme biochemical properties, low immunogenicity, and the ability to bind targets with high affinity and selectivity. While many CDPs have potential and confirmed therapeutic uses, synthesis of CDPs is a challenge. Recent advances have made the recombinant expression of CDPs a viable alternative to chemical synthesis. Moreover, identifying CDPs that can be expressed in mammalian cells is crucial in predicting their compatibility with gene therapy and mRNA therapy. Currently, we lack the ability to identify CDPs that will express recombinantly in mammalian cells without labour intensive experimentation. To address this, we developed CysPresso, a novel machine learning model that predicts recombinant expression of CDPs based on primary sequence.ResultsWe tested various protein representations generated by deep learning algorithms (SeqVec, proteInfer, AlphaFold2) for their suitability in predicting CDP expression and found that AlphaFold2 representations possessed the best predictive features. We then optimized the model by concatenation of AlphaFold2 representations, time series transformation with random convolutional kernels, and dataset partitioning.ConclusionOur novel model, CysPresso, is the first to successfully predict recombinant CDP expression in mammalian cells and is particularly well suited for predicting recombinant expression of knottin peptides. When preprocessing the deep learning protein representation for supervised machine learning, we found that random convolutional kernel transformation preserves more pertinent information relevant for predicting expressibility than embedding averaging. Our study showcases the applicability of deep learning-based protein representations, such as those provided by AlphaFold2, in tasks beyond structure prediction.

Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells.

Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep® tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.