Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

YeastFab Cloning of Toxic Genes and Protein Expression Optimization in Yeast.

YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene.

In Vitro Genome-Wide Identification of Transcription Factor Binding Sites.

DNA affinity purification followed by sequencing (DAP-seq) profiles transcription factor (TF) binding sites in vitro, in which recombinant TF captures the genomic fragments with TF binding sites. Here, I describe the preparation of pre-PCR DNA-seq libraries for 96 TFs, including the expression and purification of recombinant TF, the affinity purification of TF binding fragments, and the sequencing-ready library preparation. Using this method, up to 96 DAP-seq libraries can be prepared in 2 days.

Detection and optimization of microbial expression systems for extracellular production and purification of Ca2+-responsive phase-changing annexin fusions.

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.

Benchmarking and building DNA binding affinity models using allele-specific and allele-agnostic transcription factor binding data.

BACKGROUND: Transcription factors (TFs) bind to DNA in a highly sequence-specific manner. This specificity manifests itself in vivo as differences in TF occupancy between the two alleles at heterozygous loci. Genome-scale assays such as ChIP-seq currently are limited in their power to detect allele-specific binding (ASB) both in terms of read coverage and representation of individual variants in the cell lines used. This makes prediction of allelic differences in TF binding from sequence alone desirable, provided that the reliability of such predictions can be quantitatively assessed. RESULTS: We here propose methods for benchmarking sequence-to-affinity models for TF binding in terms of their ability to predict allelic imbalances in ChIP-seq counts. We use a likelihood function based on an over-dispersed binomial distribution to aggregate evidence for allelic preference across the genome without requiring statistical significance for individual variants. This allows us to systematically compare predictive performance when multiple binding models for the same TF are available. To facilitate the de novo inference of high-quality models from paired-end in vivo binding data such as ChIP-seq, ChIP-exo, and CUT&Tag without read mapping or peak calling, we introduce an extensible reimplementation of our biophysically interpretable machine learning framework named PyProBound. Explicitly accounting for assay-specific bias in DNA fragmentation rate when training on ChIP-seq yields improved TF binding models. Moreover, we show how PyProBound can leverage our threshold-free ASB likelihood function to perform de novo motif discovery using allele-specific ChIP-seq counts. CONCLUSION: Our work provides new strategies for predicting the functional impact of non-coding variants.

Proteomic analysis identified proteins that are differentially expressed in the flavonoid and carotenoid biosynthetic pathways of Camellia Nitidissima flowers.

BACKGROUND: Camellia nitidissima Chi is a popular ornamental plant because of its golden flowers, which contain flavonoids and carotenoids. To understand the regulatory mechanism of golden color formation, the metabolites of C. nitidissima petals at five different developmental stages were detected, a proteome map of petals was first constructed via tandem mass tag (TMT) analysis, and the accuracy of the sequencing data was validated via parallel reaction monitoring (PRM). RESULTS: Nineteen color components were detected, and most of these components were carotenoids that gradually accumulated, while some metabolites were flavonoids that were gradually depleted. A total of 97,647 spectra were obtained, and 6,789 quantifiable proteins were identified. Then, 1,319 differentially expressed proteins (DEPs) were found, 55 of which belong to the flavonoid and carotenoid pathways, as revealed by pairwise comparisons of protein expression levels across the five developmental stages. Notably, most DEPs involved in the synthesis of flavonoids, such as phenylalanine ammonium lyase and 4-coumarate-CoA ligase, were downregulated during petal development, whereas DEPs involved in carotenoid synthesis, such as phytoene synthase, 1-deoxy-D-xylulose-5-phosphate synthase, and ß-cyclase, tended to be upregulated. Furthermore, protein‒protein interaction (PPI) network analysis revealed that these 55 DEPs formed two distinct PPI networks closely tied to the flavonoid and carotenoid synthesis pathways. Phytoene synthase and chalcone synthase exhibited extensive interactions with numerous other proteins and displayed high connectivity within the PPI networks, suggesting their pivotal biological functions in flavonoid and carotenoid biosynthesis. CONCLUSION: Proteomic data on the flavonoid and carotenoid biosynthesis pathways were obtained, and the regulatory roles of the DEPs were analyzed, which provided a theoretical basis for further understanding the golden color formation mechanism of C. nitidissima.

Discovery of genes that positively affect biomass and stress associated traits in poplar.

Woody biomass serves as a renewable resource for various industries, including pulp and paper production, construction, biofuels, and electricity generation. However, the molecular mechanisms behind biomass traits are poorly understood, which significantly curtails the speed and efficiency of their improvement. We used activation tagging to discover genes that can positively affect tree biomass-associated traits. We generated and screened under greenhouse conditions a population of 2,700 independent activation tagging lines. A total of 761 lines, which had significantly and positively affected at least one biomass-associated trait, were discovered. The tag was positioned in the genome for forty lines which were affected in multiple traits and activation of proximal genes validated for a subset. For two lines we fully recapitulated the phenotype of the original lines through overexpression. Moreover, the overexpression led to more pronounced and additional improvements, not observed in the original lines. Importantly, the overexpression of a Fasciclin-like gene (PtaFLA10) and a Patatin-like gene (PtaPAT) was found to substantially improve biomass, with a 40% increase in dry-stem weight, and enhance drought tolerance, respectively. Additionally, PtaPAT overexpression increased cellulose content, which is crucial for biofuel production. Our work shows that the activation tagging approach applied even on a non-genome saturation scale in a poplar tree can be successfully used for the discovery of genes positively modify biomass productivity. Such dominant forward genetics approaches can aid in biotechnological manipulation of woody biomass traits and help unravel the functions and mechanisms of individual genes, gene families, and regulatory modules.

Quantifying and reducing the cost of tagging: combining computational fluid dynamics and diving experiments to reduce impact from animal-borne tags.

Animal-borne instruments are essential research tools for ecologists and physiologists. An increasing number of studies have shown impacts of carrying a tag on behaviour and energetics, which can have implications for animal welfare and data validity. Such impacts are a result of the additional mass and/or drag loads, with the latter requiring empirical measurements or computational fluid dynamics (CFD) to estimate. To quantify and effectively minimize tag impacts from drag, a novel combined empirical and CFD approach is required. Here, we demonstrate such an approach using captive phocid seals and the widely used Sea Mammal Research Unit (SMRU) Instrumentation Group GPS/GSM tag. We (i) show a significant change in the behaviour of grey seals when carrying a tag (gen 1; associated with 16.4% additional drag); (ii) redesigned the tag (gen 2) resulting in a lower additional drag of 8.6%; (iii) show significant differences in behaviour when carrying a gen 2 compared to gen 1 tag, demonstrating that the redesign successfully reduced impact; and (iv) observed changes in the swim speed of seals that were consistent with predictions from CFD estimates of drag. The gen 2 instrument is now commercially available. This non-trivial case study should pave the way for similar studies in other taxa and species.

Genetic Tagging and Imaging of Proteins with iFAST in Candida albicans.

Candida albicans is the most common human fungal pathogen, able to reside in a broad range of niches within the human body. Even though C. albicans systemic infection is associated with high mortality, the fungus has historically received relatively little attention, resulting in a lack of optimized molecular and fluorescent tools. Over the last decade, some extra focus has been put on the optimization of fluorescent proteins (FPs) of C. albicans. However, as the FPs are GFP-type, they require an aerobic environment and a relatively long period to fully mature. Recently, we have shown the application of a novel type of fluorogen-based FP, with an improved version of fluorescence activating and absorption shifting tag (iFAST), in C. albicans. Due to the dynamic relation between iFAST and its fluorogens, the system has the advantage of being reversible in terms of fluorescence. Furthermore, the combination of iFAST with different fluorogens results in different spectral and cellular properties, allowing customization of the system. Key features • Genetic integration and tagging with the iFAST tag in Candida albicans. • Imaging and localization of a protein of interest tagged with iFAST. • Reversibility of fluorescence with iFAST.

The role of pleiotropy and population structure in the evolution of altruism through the greenbeard effect.

Several empirical examples and theoretical models suggest that the greenbeard effect may be an important mechanism in driving the evolution of altruism. However, previous theoretical models rely on assumptions such as spatial structure and specific sets of pleiotropic loci, the importance of which for the evolution of altruism has not been studied. Here, we develop a population-genetic model that clarifies the roles of extrinsic assortment (e.g., due to population viscosity) and pleiotropy in the maintenance of altruism through the greenbeard effect. We show that, when extrinsic assortment is too weak to promote the evolution of altruism on its own, the greenbeard effect can only promote altruism significantly if there is a pleiotropic locus controlling both altruism and signaling. Further, we show that indirect selection via genetic associations is too weak to have a noticeable impact on altruism evolution. We also highlight that, if extrinsic assortment is strong enough to promote the evolution of altruism on its own, it also favors the spread of alleles encoding the other functions of a greenbeard trait (signaling and discriminatory behavior), as well as genetic associations. This occurs despite the fact that the greenbeard effect did not favor the evolution of altruism in the first place. This calls for caution when inferring the causality between greenbeard traits and the evolution of altruism.

Examining transfer of TERT to mitochondria under oxidative stress.

The primary role of telomerase is the lengthening of telomeres. Nonetheless, emerging evidence highlights additional functions of telomerase outside of the nucleus. Specifically, its catalytic subunit, TERT (Telomerase Reverse Transcriptase), is detected in the cytosol and mitochondria. Several studies have suggested an elevation in TERT concentration within mitochondria in response to oxidative stress. However, the origin of this mitochondrial TERT, whether transported from the nucleus or synthesized de novo, remains uncertain. In this study, we investigate the redistribution of TERT, labeled with a SNAP-tag, in response to oxidative stress using laser scanning fluorescence microscopy. Our findings reveal that, under our experimental conditions, there is no discernible transport of TERT from the nucleus to the mitochondria due to oxidative stress.

Tag-Array-Based UHF Passive RFID Tag Attitude Identification of Tracking Methods.

Attitude information is as important as position information in describing and localizing objects. Based on this, this paper proposes a method for object attitude sensing utilizing ultra-high frequency passive RFID technology. This method adopts a double tag array strategy, which effectively enhances the spatial freedom and eliminates phase ambiguity by leveraging the phase difference information between the two tags. Additionally, we delve into the issue of the phase shift caused by coupling interference between the two tags. To effectively compensate for this coupling effect, a series of experiments were conducted to thoroughly examine the specific impact of coupling effects between tags, and based on these findings, a coupling model between tags was established. This model was then integrated into the original phase model to correct for the effects of phase shift, significantly improving the sensing accuracy. Furthermore, we considered the influence of the object rotation angle on phase changes to construct an accurate object attitude recognition and tracking model. To reduce random errors during phase measurement, we employed a polynomial regression method to fit the measured tag phase information, further enhancing the precision of the sensing model. Compared to traditional positioning modes, the dual-tag array strategy essentially increases the number of virtual antennas available for positioning, providing the system with more refined directional discrimination capabilities. The experimental results demonstrated that incorporating the effects of inter-tag coupling interference and rotation angle into the phase model significantly improved the recognition accuracy for both object localization and attitude angle determination. Specifically, the average error of object positioning was reduced to 12.3 cm, while the average error of attitude angle recognition was reduced to 8.28°, making the method suitable for various practical application scenarios requiring attitude recognition.

Vitamin D Significantly Inhibits Carcinogenesis in the Mogp-TAg Mouse Model of Fallopian Tube Ovarian Cancer.

Epidemiological and observational studies suggest that vitamin D has potential for the chemoprevention of ovarian cancer. The anticancer effect of vitamin D in the fallopian tube epithelium (FTE), which is now thought to harbor the precursor cells for high grade ovarian cancer, is not known. The purpose of this study was to investigate whether vitamin D can inhibit carcinogenesis in the mogp-TAg fallopian tube (FT) ovarian cancer mouse model and examine underlying mechanisms. To test this hypothesis, 3 groups of 40 5-week-old female mogp-TAg mice were divided equally into two cohorts of 20 mice, treated with either vehicle (vitamin D solvent) or the active 1,25(OH)2D3 analogue EB1089, delivered via mini-pump or IP injection or cholecalciferol delivered in the feed. The FTs were characterized histologically and pathologically after 3 and 7 weeks of treatment. The effect of vitamin D on cultured human FTE cells was also examined. After 3 weeks, vitamin D, delivered as either cholecalciferol or EB1089 significantly inhibited FT carcinogenesis. After 7 weeks, cholecalciferol significantly reduced p53 signatures, serous tubal epithelial carcinoma, FT cancer, and plasma CA125 while increasing apoptosis in the FTE. EB1089 had no significant effect on FT carcinogenesis at 7 weeks. Cholecalciferol significantly reduced proliferation and increased apoptosis in vitro in p53-altered FTE cells. In conclusion, vitamin D inhibited FT carcinogenesis by clearing cells with p53 alterations. These data suggest that vitamin D has merit for the chemoprevention of fallopian tube/ovarian cancer. The optimal chemopreventive effect may be dependent on the route of vitamin D administration.

Evidence for Greater Marking along Ethnic Boundaries.

The coordination of beliefs, norms, and behaviors is foundational to theories of group formation. However, because beliefs and norms are not directly observable, signaling mechanisms are required to build reliable signals of latent traits. Although the mathematical theory behind these signals is robust, there is very little testing of ethnic marker theory or of its key propositions that markers become more prevalent along ethnic boundaries and where more than two cultural groups are in contact. We present an ethnographic test of this theory with phonetic differences serving as potential group signals. The data derive from an ethnographic and linguistic investigation in two contrasting secondary school settings in Utah, one that is majority European American and one that is ethnically more diverse. Word list recordings were collected as part of interviews with teens from different backgrounds. We extracted acoustic data from the speech of European Americans (EAs) and Pacific Islanders (PIs), then analyzed differences in the pronunciation of the vowel in words such as "bit." We found evidence of greater phonetic marking at the more diverse school, along more prominent boundaries of ethnic interaction. These results align with predictions made by the theoretical models. This initial empirical test of model predictions provides justification for the development of more complex models that could account for more variables within the environment.

The concentration of free glycerol in goat milk increases during feed restrictions.

This Research Communication introduces a novel enzymatic-fluorometric analytical procedure for glycerol and glycerol 3-phosphate in milk. Milk from thirty-seven goats was analysed during 9 consecutive days during which a two-day feed restriction was introduced. Fractional milk triacylglyceride and free glycerol increased significantly while glycerol 3-phosphate reacted more moderately. The energy status of the mammary cell is discussed.

Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen.

Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determination of cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0 pg·mL-1, representing two orders of magnitude improvement compared with equivalent microtiter plate-based assay (LOD of 460 pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.

Enhancer looping protein LDB1 modulates MYB expression in T-ALL cell lines in vitro by cooperating with master transcription factors.

BACKGROUND: Despite significant progress in the prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) in recent decades, a notable portion of children still confronts challenges such as treatment resistance and recurrence, leading to limited options and a poor prognosis. LIM domain-binding protein 1 (LDB1) has been confirmed to exert a crucial role in various physiological and pathological processes. In our research, we aim to elucidate the underlying function and mechanisms of LDB1 within the background of T-ALL. METHODS: Employing short hairpin RNA (shRNA) techniques, we delineated the functional impact of LDB1 in T-ALL cell lines. Through the application of RNA-Seq, CUT&Tag, and immunoprecipitation assays, we scrutinized master transcription factors cooperating with LDB1 and identified downstream targets under LDB1 regulation. RESULTS: LDB1 emerges as a critical transcription factor co-activator in cell lines derived from T-ALL. It primarily collaborates with master transcription factors (ERG, ETV6, IRF1) to cooperatively regulate the transcription of downstream target genes. Both in vitro and in vivo experiments affirm the essential fuction of LDB1 in the proliferation and survival of cell lines derived from T-ALL, with MYB identified as a significant downstream target of LDB1. CONCLUSIONS: To sum up, our research establishes the pivotal fuction of LDB1 in the tumorigenesis and progression of T-ALL cell lines. Mechanistic insights reveal that LDB1 cooperates with ERG, ETV6, and IRF1 to modulate the expression of downstream effector genes. Furthermore, LDB1 controls MYB through remote enhancer modulation, providing valuable mechanistic insights into its involvement in the progression of T-ALL.

Snorkel-tag based affinity chromatography for recombinant extracellular vesicle purification.

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine is still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present the Snorkel-tag, for which we have genetically fused the EV surface marker protein CD81, to a series of tags with an additional transmembrane domain to be displayed on the EV surface, resembling a snorkel. This system enables the affinity purification of EVs from complex matrices in a non-destructive form while maintaining EV characteristics in terms of surface protein profiles, associated miRNA patterns and uptake into a model cell line. Therefore, we consider the Snorkel-tag to be a widely applicable tool in EV research, allowing for efficient preparation of EV standards and reference materials, or dissecting EVs with different surface markers when fusing to other tetraspanins in vitro or in vivo.

Characterization of Ksg1 protein kinase-dependent phosphoproteome in the fission yeast S. pombe.

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

A SAW Wireless Passive Sensing System for Rotating Metal Parts.

Passive wireless surface acoustic wave (SAW) sensors are very useful for on-site monitoring of the working status of machines in complex environments, such as high-temperature rotating objects. For rotating parts, it is difficult to realize real-time and continuous monitoring because of the unstable sensing signal caused by the continuous change of the relative position of the rotating part to the sensor and shielding of the signal. In our SAW sensing system, we propose a loop antenna integrated with the rotating part to obtain a stable sensing signal owing to its omnidirectional radiation pattern. Methodologies for determining the antenna dimension, system operating frequency, and procedures for designing a SAW sensor tag are discussed in this paper. By fully utilizing the influence of metal rotor on antenna performance, the antenna needs no impedance matching elements while it provides sufficient gain, which equips the antenna with nearly zero temperature drift at a wide temperature-sensing range. Experimental verification results show that this sensing system can greatly improve the stability of the sensing signal significantly and can achieve a temperature sensing accuracy of ~1 °C at different rotational speeds, demonstrated by the feasibility of the loop antenna for monitoring the working status of rotating metal parts.