微生物学

Nucleases are key tools in molecular biology, enabling controlled nucleic acid digestion for applications such as ribosome profiling. Micrococcal nuclease (MNase) from Staphylococcus aureus is widely used as a tool in molecular biology and biochemistry, but its reduced activity under high-salt conditions necessitates higher enzyme input to achieve efficient digestion, increasing costs in studies of halophilic organisms. Here, we present an optimized protocol for the heterologous expression and purification of the recombinant staphylococcal MNase. The procedure enables reproducible production of a highly active, stable enzyme and incorporates an enzymatic activity assay to standardize batches to minimize variability. The resulting MNase exhibits robust activity in high-salt environments and remains stable during storage, providing a cost-effective and reliable alternative to commercial nucleases for ribosome profiling and related applications.

The CRISPR-Cas system of Thermus thermophilus has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools.

Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from Acinetobacter genomospecies 16 (A. gp16), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments.

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.

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

Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain–only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification.

Key features

• Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment.

• Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.

Graphical overview