往期刊物2024
卷册: 14, 期号: 7
生物化学
In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase
内部提取和纯化Pfu-Sso7d：一种高持续性的DNA聚合酶
The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase.Key features• We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d.• Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance.• Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers.• The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCRapplications.
Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin
使用丹磺酰化钙调蛋白的荧光测定法测量钙调素依赖性肽-蛋白相互作用
The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions.Key features• Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding.• Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts.• Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development.• Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.Graphical overviewFluorescence emission profiles of dansylated-calmodulin (D-CaM) in different states. Fluorescence emission spectra of D-CaM upon excitation at 320 nm are depicted. Conditions include apo-D-CaM (gray), holo-D-CaM (red), apo-D-CaM bound to peptide (blue), and holo-D-CaM bound to peptide (purple). Corresponding structural representations of D-CaM next to each condition are superimposed on the respective spectra along with the hydrophobicity of the dansyl environment, which increases upon binding of peptide or Ca2+ to D-CaM. Upon peptide binding to D-CaM, there is an enhancement in the fluorescent intensity of the spectra; upon Ca2+ binding, there is an enhancement of the intensity and a leftward shift of the spectra.
生物工程
Method for Large-scale Production of hIPSC Spheroids
大规模生产hIPSC球体的方法
Stem cell spheroids are rapidly becoming essential tools for a diverse array of applications ranging from tissue engineering to 3D cell models and fundamental biology. Given the increasing prominence of biotechnology, there is a pressing need to develop more accessible, efficient, and reproducible methods for producing these models. Various techniques such as hanging drop, rotating wall vessel, magnetic levitation, or microfluidics have been employed to generate spheroids. However, none of these methods facilitate the easy and efficient production of a large number of spheroids using a standard 6-well plate. Here, we present a novel method based on pellet culture (utilizing U-shaped microstructures) using a silicon mold produced through 3D printing, along with a detailed and illustrated manufacturing protocol. This technique enables the rapid production of reproducible and controlled spheroids (for 1 × 106 cells, spheroids = 130 ± 10 μm) from human induced pluripotent stem cells (hIPSCs) within a short time frame (24 h). Importantly, the method allows the production of large quantities (2 × 104 spheroids for 1 × 106 cells) in an accessible and cost-effective manner, thanks to the use of a reusable mold. The protocols outlined herein are easily implementable, and all the necessary files for the method replication are freely available.Key features• Provision of 3D mold files (STL) to produce silicone induction device of spheroids using 3D printing.• Cost-effective, reusable, and autoclavable device capable of generating up to 1.2× 104 spheroids of tunable diameters in a 6-well plate.• Spheroids induction with multiple hIPSC cell lines.• Robust and reproducible production method suitable for routine laboratory use.Graphical overviewSpheroid induction process following the pellet method on molded silicon discs
微生物学
Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins
从大肠杆菌中纯化发光杆菌毒力盒（PVC）蛋白复合物，用于异源货物蛋白的人工转运
Contractile injection systems (CISs), one of the most important bacterial secretion systems that transport substrates across the membrane, are a collection of diverse but evolutionarily related macromolecular devices. Numerous effector proteins can be loaded and injected by this secretion complex to their specific destinations. One group of CISs called extracellular CIS (eCIS) has been proposed as secretory molecules that can be released from the bacterial cytoplasm and attack neighboring target cells from the extracellular environment. This makes them a potential delivery vector for the transportation of various cargos without the inclusion of bacterial cells, which might elicit certain immunological responses from hosts. We have demonstrated that the Photorhabdus virulence cassette (PVC), which is a typical eCIS, could be applied as an ideal vector for the translocation of proteinaceous cargos with different physical or chemical properties. Here, we describe the in-depth purification protocol of this mega complex from Escherichia coli. The protocol provided is a simpler, faster, and more productive way of generating the eCIS complexes than available methodologies reported previously, which can facilitate the subsequent applications of these nanodevices and other eCIS in different backgrounds.
Purification of Native Dentilisin Complex from Treponema denticola by Preparative Continuous Polyacrylamide Gel Electrophoresis and Functional Analysis by Gelatin Zymography
通过制备连续聚丙烯酰胺凝胶电泳和凝胶酶谱分析法纯化来自梭状龋齿螺旋体的天然Dentilisin复合物及其功能分析
Periodontal disease is characterized by the destruction of the hard and soft tissues comprising the periodontium. This destruction translates to a degradation of the extracellular matrices (ECM), mediated by bacterial proteases, host-derived matrix metalloproteinases (MMPs), and other proteases released by host tissues and immune cells. Bacterial pathogens interact with host tissue, triggering adverse cellular functions, including a heightened immune response, tissue destruction, and tissue migration. The oral spirochete Treponema denticola is highly associated with periodontal disease. Dentilisin, a T. denticola outer membrane protein complex, contributes to the chronic activation of pro-MMP-2 in periodontal ligament (PDL) cells and triggers increased expression levels of activators and effectors of active MMP-2 in PDL cells. Despite these advances, no mechanism for dentilisin-induced MMP-2 activation or PDL cytopathic behaviors leading to disease is known. Here, we describe a method for purification of large amounts of the dentilisin protease complex from T. denticola and demonstrate its ability to activate MMP-2, a key regulator of periodontal tissue homeostasis. The T. denticola dentilisin and MMP-2 activation model presented here may provide new insights into the dentilisin protein and identify potential therapeutic targets for further research.
神经科学
Nerve Preparation and Recordings for Pharmacological Tests of Sensory and Nociceptive Fiber Conduction Ex Vivo
药理学测试中感觉和伤害感受神经纤维传导的离体神经准备与记录
Measuring signal propagation through nerves is a classical electrophysiological technique established decades ago to evaluate sensory and motor functions in the nervous system. The whole-nerve preparation provides a valuable model to investigate nerve function ex vivo; however, it requires specific knowledge to ensure successful and stable measurements. Although the methodology for sciatic nerve recordings has long existed, a method for reliable and long-lasting recordings from myelinated and non-myelinated (nociceptive) fibers still needs to be adapted for pharmacological testing. This protocol takes benefits from epineurium sheath removal for pharmacological tests and provides a detailed description of how to make accurate nerve preparations, from the dissection and handling of nerves to epineurium cleaning, fabrication of adaptable suction electrodes for appropriate fiber stimulation and recordings, setting of electrophysiological protocols for compound action potential (CAP) recordings to distinguish between myelinated and non-myelinated (nociceptive) fibers, and finally to the analysis of the datasets of CAP components. We also demonstrate the feasibility of CAP recordings from individual branches in epineurium-free nerve preparations and provide clues to help retain nerve viability and maintain stable recordings over time. Although a sciatic nerve preparation was used here, the methodology can be applied to other nerve-type preparations.Key features• Detailed and simplified protocol for peripheral nerve preparation for recording sensory inputs ex vivo.• Recordings from myelinated and non-myelinated (nociceptive) fibers can be performed hours after nerve preparation.• The protocol involves the epineurium removal to facilitate drug permeability into nerve tissue for pharmacological tests.• The protocol allows physiological and pathological studies (pain/chronic pain conditions).Graphical overview Preparation and recordings from the sciatic nerve, including myelinated and non-myelinated (nociceptive) fibers
植物科学
Agrobacterium-Mediated Transient Gene Expression Optimized for the Bioenergy Crop Camelina sativa
优化农杆菌介导的生物能源作物亚麻籽荠瞬时性基因表达
Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1–32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system.Key features• This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop Camelina species.• A constitutive and inducible transient gene expression in the hexaploid species Camelina sativa cultivar DH55.• Requires only 16–18 days to complete with high efficacy.Graphical overviewAgrobacterium-mediated transient gene expression optimized for Camelina sativa
Transient Expression Assay and Microscopic Observation in Kumquat Fruit
金桔果实瞬时表达测定与显微观察
Citrus fruits encompass a diverse family, including oranges, mandarins, grapefruits, limes, kumquats, lemons, and others. In citrus, Agrobacterium tumefaciens–mediated genetic transformation of Hongkong kumquat (Fortunella hindsii Swingle) has been widely employed for gene function analysis. However, the perennial nature of woody plants results in the generation of transgenic fruits taking several years. Here, we show the procedures of Agrobacterium-mediated transient transformation and live-cell imaging in kumquat (F. crassifolia Swingle) fruit, using the actin filament marker GFP-Lifeact as an example. Fluorescence detection, western blot analysis, and live-cell imaging with confocal microscopy demonstrate the high transformation efficiency and an extended expression window of this system. Overall, Agrobacterium-mediated transient transformation of kumquat fruits provides a rapid and effective method for studying gene function in fruit, enabling the effective observation of diverse cellular processes in fruit biology.