Protein Expression and Purification

Efficient production of human proteins for the development of tool compounds and biologics depends on a detailed understanding of the protein expression machinery in mammalian cells. Codon optimization is widely believed to enhance protein yield, yet its impact in homologous mammalian systems remains poorly defined. Here, we systematically compare five codon usage strategies reflecting common assumptions about rare codons, RNA stability, and synthesis efficiency. We developed pTipi, an efficient open-source mammalian expression vector, and evaluated its performance in antibody production. We generated plasmids for common epitope tag antibodies such as V5, anti-biotin and anti-His for distribution by Addgene. To compare codon usage schemes, we performed a bake-off of 18 human and murine Wnt pathway glycoproteins in mammalian cells. Small-scale expression screens revealed that codon optimization did not provide a general advantage over native coding sequences, while strategies prioritizing RNA stability consistently reduced expression. Interestingly, a skewed codon scheme using the most abundant codons produced yields comparable to native sequences and occasionally enhanced protein output. To enable flexible evaluation of codon strategies, we implemented a Golden Gate-compatible pTipi platform for efficient synthetic gene incorporation. We conclude that native codons are sufficient for robust homologous mammalian expression of glycoproteins, while selective codon skewing can be beneficial for some targets.

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduction in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability assays. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold increase in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

RNase R is a processive 3 to 5 exoribonuclease that degrades a broad array of linear RNA species while preserving RNA lariats and circular RNAs (circRNA). In recent years, this enzyme has become pivotal for the field of circRNA research, serving as a key step for circRNA enrichment, purification, and identification. Despite this growing importance, the effects of mutations and truncations in RNase R have been incompletely studied. We make several point mutations and assay their effects on the ability of RNase R to bind and/or degrade RNA substrates. Our data show that selected active site mutations have varying effects on RNA binding and degradation. Furthermore, the increasing interest in circRNA-based RNA therapeutic platforms highlights an urgent need for RNase R in RNA molecular biology labs. However, the substantial cost of commercial RNase R remains a bottleneck, particularly for large-scale studies or the development of circRNA-based technologies. In this protocol, we offer a solution to that problem, namely a more accessible and cost-effective means of purifying high-quality and low-cost RNase R. We provide a highly detailed yet simplified, high-yield protocol that produces recombinant RNase R from Escherichia coli. The method uses a single-step Ni-NTA affinity chromatography procedure without proteolytic tag removal and is optimized for entry-level FPLC systems such as the AKTA Start, ensuring that high-purity enzyme production does not require specialized, high-end instrumentation. A second key feature is the establishment of an optimized reaction framework, including specific buffer compositions and defined enzyme-to-substrate ratios for the purified RNase R. The protocol achieves functional equivalence to premium commercial RNase R, ensuring complete linear RNA digestion without compromising the integrity of circRNA. The combination of a simplified purification workflow and a robust reaction protocol provides an accessible, cost-effective, and reliable solution for any molecular biology laboratory requiring high volumes of RNase R. Key FeaturesO_LIRNase R mutations can block RNase activity, RNA binding or both C_LIO_LIThis protocol purifies [~]40 mg of active RNase R per liter of E. coli culture C_LIO_LIThe protocol avoids medium and high end FPLC systems C_LIO_LIRNase R expression constructs (WT and mutants) will be available on Addgene C_LIO_LIThe protocol includes an optimized reaction buffer to pair with this RNase R C_LIO_LIOptional endotoxin removal step is also included C_LI

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

Recombinant peptide production was pioneered in the 1970s for the generation of therapeutic peptides, with notable examples including insulin and somatostatin. These early methods required the use of cyanogen bromide (BrCN) for cleavage of the native peptide sequence from a fusion protein. Since that time, while numerous BrCN-dependent peptide methods continue to be reported, the accessibility and cost of site-specific proteases have improved dramatically. These developments have enabled alternative approaches to recombinant peptide generation that obviate the need for BrCN, an environmentally destructive toxin. We recently created an immobilized SUMO protease that can replace BrCN usage in recombinant peptide production workflows by releasing native peptides expressed as part of a SUMO-peptide fusion protein. We have used this approach to generate P113 peptide, the minimal active fragment of the antifungal peptide Histatin 5. In this report, we describe the creation and characterization of this immobilized SUMO protease and its application in the production of experimentally viable quantities of active P113 peptide.

Quality and price of fetal bovine serum (FBS) are traditionally determined by geographical origin and parameters listed in the Certificate of Analysis (CoA). Despite its central role in cell culture, selecting suitable FBS batches remains costly and labor-intensive due to substantial batch-to-batch variation. We propose a molecular assessment strategy based on transcriptomic and cytokine profiling of cells cultured in different FBS batches to evaluate performance more reliably. Analysis of differential gene expression in three cell lines - MRC-5, Jurkat, and THP-1 - enables batch grouping and reveals pathway-specific effects, with immune-related pathways showing the most pronounced variability. Although CoA parameters can stratify batches by origin, they do not consistently correlate with cytokine secretion or gene expression across cell lines. These findings demonstrate that geographical origin is an inadequate predictor of functional FBS performance and that molecular profiling provides a more robust and informative assessment.

BackgroundShigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo, to generate a "double-hit" Shigella glycoconjugate vaccine. MethodGlycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. ResultsWhen co-produced in E. coli, PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella-specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. ConclusionA novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a "double-hit", immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

The ubiquitin-conjugating enzyme UBE2J1 functions in the proteasomal degradation of proteins at the ER. Existing evidence suggests that it plays a role during viral infection, with elevated UBE2J1 levels generally associated with increased infection. This is particularly relevant for some RNA viruses; however, the regulation of UBE2J1 during infection has not been well studied. Here, we used a BHK21 cell model to demonstrate that UBE2J1 overexpression promotes the replication of Vesicular Stomatitis Virus (VSV), as indicated by a significant increase in viral titres. To better understand the underlying molecular processes, cells were co-transfected to express the VSV-G protein and wild-type UBE2J1 protein, and we observed a significant increase in the syncytial fusion area. This effect was not observed when catalytically inactive (C91S) or phospho-deficient (S184A) forms of the protein were used. Interestingly, overexpression of a truncated, non-ER localized form of UBE2J1 ({Delta}TM) led to the largest increase in the syncytial fusion area. This arose as a result of increased syncytia size, and may indicate an enhanced cellular role if soluble forms of UBE2J1 are not anchored to the ER. Additional studies using truncated, mutated and wild-type proteins confirmed that UBE2J1 increases VSV viral replication and is associated with an increase in the number of infection plaques. Considering the emerging evidence for UBE2J1 involvement in viral infection, our finding should help in understanding the role of this protein in viral pathogenesis and cellular processes linked to syncytialization.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Myofibrillar proteins, namely actin and myosin, are responsible for many of the textural attributes of animal-based meat. Precision fermentation (recombinant production of food ingredients) represents an underexplored approach to producing these proteins without the unsustainable practice of animal agriculture. We show that through the solubility-enhancing SUMO peptide tag and precipitation-based purification, we can produce actin via recombinant DNA methods at titers of 326 mg/L E. coli culture. We also show expression and precipitation of a recombinant fragment of the myosin tail, leading to 572 mg/L culture. For both proteins, yields are improved compared to prior studies, without the need for low-yielding laborious purification columns, with final purities of 69-73%. These recombinant actin and myosin proteins showed macro- and microscopic fibrous features similar to meat. When combined with plant-based proteins, chewiness, hardness, and Youngs modulus were improved towards that of animal-based meat. Preliminary cost analyses suggest a less expensive process for producing myofibrillar proteins compared to established methods. Our results reveal a novel scalable approach to making meat-like foods and ingredients through precision fermentation.

BackgroundSuccinic acid (SA) is a four-carbon dicarboxylic acid of considerable industrial relevance, with applications spanning the food, chemical, and pharmaceutical sectors. The remarkable acid tolerance of the yeast Yarrowia lipolytica makes it a promising microbial cell factory for SA production. Numerous metabolic engineering strategies have focused on disrupting genes encoding the succinate dehydrogenase (SDH) complex to enhance SA accumulation. However, such a modification is associated with impaired growth and the accumulation of by-products, notably acetic acid (AA). ResultsTo improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5-deficient strains (Sdh5{Delta}), carbon flux from glycolysis was partially redirected toward the pentose phosphate pathway by overexpression of the native genes encoding glucose-6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), thereby enhancing NADPH generation. The resulting strain was further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase (TfMdh) from Thermus flavus or the soluble transhydrogenase (EcSthA) from Escherichia coli, enabling indirect conversion of NADPH to NADH. This strategy resulted in 2-fold and 2.2-fold increase in SA productivity and titre, respectively, compared to the Sdh5{Delta}-ALE strain during bioreactor cultivation on glucose-based media. Moreover, AA accumulation was reduced 1.2-fold, while growth rates were significantly improved. ConclusionsThe proposed engineering strategies, especially heterologous expression of EcSthA, partly alleviated energy limitations in Y. lipolytica Sdh5{Delta} strain, resulting in improved SA productivity and growth performance.

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1457b00org.highwire.dtl.DTLVardef@8e7405org.highwire.dtl.DTLVardef@6303eforg.highwire.dtl.DTLVardef@974d71_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

Natural extracts could improve sperm storage and artificial insemination (AI). This study, for the first time, evaluates the suitability of a blueberry extract (Vaccinium corymbosum) obtained from pomace using a sustainable methodology as a supplement for bull semen extenders. Cryopreserved semen doses from eight bulls were combined in 9 pools (3 bulls/pool), supplemented with 0%, 1%, 5%, or 10% extract, and incubated up to 5 h at 38 {degrees}C. Motility was assessed hourly using OpenCASA, and the effects of treatment and time were evaluated using linear mixed-effects models. Motility was significantly better preserved with 1% extract (total and progressive motility, improved linear velocity and linearities, and decreased BCF and fractal dimension, related to hyperactivation). The effect of 5% was overall positive, but it was below 1%, whereas 10% mostly showed a negative effect. These results show that this natural extract could safely supplement bull semen extenders at least between 1% to 5%, and even help improve sperm motility. Therefore, this extract offers an opportunity to enhance cattle semen extenders using a sustainable approach, potentially improving reproductive outcomes.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a predictive indicator of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, B4GALT1, ST3GAL4, ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tuneable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited predictive power for galactosylation and 2,3-sialylation, highlighting glycosylation pathway redundancy and context dependence. This study provides the first systematic, cross-cell line evaluation of lectin-based cell-surface glycan profiling as a predictor of mAb product glycosylation, establishing its practical utility and inherent limitations for CHO glycoengineering workflows. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/724788v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@6d5cfborg.highwire.dtl.DTLVardef@1f38e0aorg.highwire.dtl.DTLVardef@f25fa2org.highwire.dtl.DTLVardef@64a0dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

The endoplasmic reticulum (ER)-Golgi interface is a dynamic trafficking hub maintained in part by TANGO1, a scaffolding protein that coordinates proteins and membranes at ER exit sites (ERES). TANGO1 has two isoforms: TANGO1L, which has a lumenal SH3 domain, and TANGO1S, which lacks this domain but retains the transmembrane and cytoplasmic coiled-coil (CC), TEER, and PRD domains common to both forms. We showed previously that loss of both isoforms disrupts ER-Golgi organization more severely than TANGO1L loss alone, indicating TANGO1S is functional and can compensate. Here we dissect the role of each TANGO1 cytoplasmic domain in maintaining secretory pathway organisation by expressing TANGO1S domain-deletion mutants in TANGO1L-/S-knockout cells. We show that TANGO1 loss causes cis-Golgi vesiculation that cannot be rescued by TANGO1S, suggesting the lumenal domain of TANGO1L is essential in supporting Golgi architecture. Meanwhile, the TEER domain is essential for the organisation of the ER, whilst the TEER, CC2 and PRD domain are required for a defined ERGIC. All constructs partially rescue COPII recruitment. This study represents an advance towards a domain-level resolution of TANGO1S function. Summary statementIn this study we perform rescue experiments in TANGO1 knockout cells to dissect the role of the TANGO1 cytoplasmic domains in maintaining the ER-ERGIC-Golgi continuum.