Protocols in Current Issue
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0 Q&A 438 Views Apr 5, 2024

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 PCR applications.

0 Q&A 248 Views Mar 20, 2024

Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify β-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes β-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration(charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC–MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species.

Key features

• Uses basic materials and laboratory equipment, enabling low-cost studies.

• Facilitates the selection and identification of proteases with certain molecular weights.

• Enables further functional studies with host proteins, such as structural or immune response–related, or enzymes and candidate protease inhibitors(e.g., from natural substances).

• This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.

Graphical overview

0 Q&A 538 Views Jan 5, 2024

Tears contain numerous secreted factors, enzymes, and proteins that help in maintaining the homeostatic condition of the eye and also protect it from the external environment. However, alterations to these enzymes and/or proteins during pathologies such as mechanical injury and viral or fungal infections can disrupt the normal ocular homeostasis, further contributing to disease development. Several tear film components have a significant role in curbing disease progression and promoting corneal regeneration. Additionally, several factors related to disease progression are secreted into the tear film, thereby serving as a valuable reservoir of biomarkers. Tears are readily available and can be collected via non-invasive techniques or simply from contact lenses. Tears can thus serve as a valuable and easy source for studying disease-specific biomarkers. Significant advancements have been made in recent years in the field of tear film proteomics, lipidomics, and transcriptomics to allow a better understanding of how tears can be utilized to gain insight into the etiology of diseases. These advancements have enabled us to study the pathophysiology of various disease states using tear samples. However, the mechanisms by which tears help to maintain corneal homeostasis and how they are able to form the first line of defense against pathogens remain poorly understood and warrant detailed in vitro studies. Herein, we have developed an in vitro assay to characterize the functional importance of patient isolated tears and their components on corneal epithelial cells. This novel approach closely mimics real physiological conditions and could help the researchers gain insight into the underlying mechanisms of ocular pathologies and develop new treatments.

Key features

• This method provides a new technique for analyzing the effect of tear components on human corneal epithelial cells.

• The components of the tears that are altered in response to diseases can be used as a biomarker for detecting ocular complications.

• This procedure can be further employed as an in vitro model for assessing the efficacy of drugs and discover potential therapeutic interventions.

0 Q&A 520 Views Jan 5, 2024

Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12–ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout.

Key features

• Preparation of ATG7 E1 from insect cells (Sf9 cells).

• Preparation of ATG3 E2 from bacteria (E. coli).

• Preparation of LC3B S3C from bacteria (E. coli).

• Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.

Graphical overview

Experimental design to track the full reaction of ATG8 lipidation, described in this protocol

0 Q&A 767 Views Dec 5, 2023

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein.

Key features

• This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works.

• The protocol requires several days to complete as various steps are designed to be performed overnight.

• The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

0 Q&A 407 Views Dec 5, 2023

Eukaryotic cells rely on actin to support cellular structure, motility, transport, and a wide variety of other cytoplasmic functions and nuclear activities. Humans and other mammals express six closely related isoforms of actin, four of which are found primarily in skeletal, cardiac, and smooth muscle tissues. The final two isoforms, β and γ, are found in non-muscle cells. Due to the ease of purification, many biochemical studies surveying the functions of actin and its regulators have been carried out with protein purified from skeletal muscle. However, it has become increasingly clear that some activities are isoform specific, necessitating more accessible sources of non-muscle actin isoforms. Recent innovations permit the purification of non-muscle actins from human cell culture and heterologous systems, such as insect cell culture and the yeast Pichia pastoris. However, these systems generate mixtures of actin types or require additional steps to remove purification-related tags. We have developed strains of Saccharomyces cerevisiae (budding yeast) that express single untagged isoforms of either human non-muscle actin (β or γ) as their sole actin, allowing the purification of individual homogeneous actin isoforms by conventional purification techniques.

Key features

• Easy growth of yeast as a source of human cytoplasmic actin isoforms.

• Uses well-established actin purification methods.

• The tag-free system requires no post-purification processing.

Graphical overview

Isolating human cytoplasmic actins from yeast

0 Q&A 486 Views Aug 5, 2023

The chloroplast lumen contains at least 80 proteins whose function and regulation are not yet fully understood. Isolating the chloroplast lumen enables the characterization of the lumenal proteins. The lumen can be isolated in several ways through thylakoid disruption using a Yeda press or sonication, or through thylakoid solubilization using a detergent. Here, we present a simple procedure to isolate thylakoid lumen by sonication using leaves of the plant Arabidopsis thaliana. The step-by-step procedure is as follows: thylakoids are isolated from chloroplasts, loosely associated thylakoid surface proteins from the stroma are removed, and the lumen fraction is collected in the supernatant following sonication and centrifugation. Compared to other procedures, this method is easy to implement and saves time, plant material, and cost. Lumenal proteins are obtained in high quantity and purity; however, some stromal membrane–associated proteins are released to the lumen fraction, so this method could be further adapted if needed by decreasing sonication power and/or time.

1 Q&A 412 Views Jul 5, 2023

Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.

Graphical overview

Workflow for autolysin production from Chlamydomonas reinhardtii

0 Q&A 366 Views Apr 5, 2023

Zebrafish is an excellent model to study vertebrate neurobiology, but its synaptic components that mediate and regulate fast electrical synaptic transmission are largely unidentified. Here, we describe methods to solubilize and immunoprecipitate adult zebrafish brain homogenate under conditions to preserve electrical synapse protein complexes. The methods presented are well-suited to probe electrical synapse immunocomplexes, and potentially other brain-derived immunocomplexes, for candidate interactors from zebrafish brain.

0 Q&A 1130 Views Mar 5, 2023

Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia’s low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach.

Graphical abstract

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