生物化学

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.

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.

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

While site-specific translational encoding of phosphoserine (pSer) into proteins in Escherichia coli via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable E. coli GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH₂) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a Streptomyces biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This “PermaPhos” expression system uses the E. coli BL21(DE3) ∆serC strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the Streptomyces nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in E. coli are particularly useful for discovering phosphorylation-dependent protein–protein interaction networks from cell lysates or transfected cells.

Key features

• Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway.

• Protein expression uses standard Terrific Broth (TB) media and requires three days to complete.

• C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein.

• Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.

Graphical overview

Nucleic acids in living organisms are more complex than the simple combinations of the four canonical nucleotides. Recent advances in biomedical research have led to the discovery of numerous naturally occurring nucleotide modifications and enzymes responsible for the synthesis of such modifications. In turn, these enzymes can be leveraged towards toolkits for DNA and RNA manipulation for epigenetic sequencing or other biotechnological applications. Here, we present the protocol to obtain purified 5-hydroxymethylcytosine carbamoyltransferase enzymes and the associated assays to convert 5-hydroxymethylcytosine to 5-carbamoyloxymethylcytosine in vitro. We include detailed assays using DNA, RNA, and single nucleotide/deoxynucleotide as substrates. These assays can be combined with downstream applications for genetic/epigenetic regulatory mechanism studies and next-generation sequencing purposes.

The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural analysis of VHH-RBD complexes by X-ray crystallography enables VHH epitopes to be precisely mapped, and the effect of variant mutations to be interpreted and predicted. Key to this is a protocol for the reproducible production and crystallization of the VHH-RBD complexes. Based on our experience, we describe a workflow for expressing and purifying the proteins, and the screening conditions for generating diffraction quality crystals of VHH-RBD complexes. Production and crystallization of protein complexes takes approximately twelve days, from construction of vectors to harvesting and freezing crystals for data collection.

Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDH_Tt from Thermus thermophilus, as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles.

In this protocol, we describe ALDH_Tt heterologous expression in E. coli, purification, and activity assay (based on Shortall et al., 2021). ALDH_Tt was first copurified as a contaminant during caa₃-type cytochrome oxidase isolation from T. thermophilus. This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15–20 mg/L ALDH_Tt. For purification of the thermophilic his-tagged ALDH_Tt, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH).

Graphical abstract:

Flow chart outlining the steps in ALDH_Tt expression and purification, highlighting the approximate time required for each step.

The human proteins used in most biochemical studies are commonly obtained using bacterial expression. Owing to its relative simplicity and low cost, this approach has been extremely successful, but is inadequate for many proteins that require the mammalian folding machinery and posttranslational modifications (PTMs) for function. Moreover, the expressed proteins are typically purified using N- and/or C-terminal affinity tags, which are often left on proteins or leave non-native extra amino acids when removed proteolytically. Many proteins cannot tolerate such extra amino acids for function. Here we describe a protein production method that resolves both these issues. Our method combines expression in human Expi293F cells, which grow in suspension to high density and can process native PTMs, with a chitin-binding domain (CBD)-intein affinity purification and self-cleavable tag, which can be precisely removed after purification. In this protocol, we describe how to clone a target gene into our specifically designed human cell expression vector (pJCX4), and how to efficiently transfect the Expi293F cells and purify the expressed proteins using a chitin affinity resin.

Graphic abstract:

Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used as a paradigm to elucidate the mechanisms of DNA binding and cleavage. Since these enzymes modulate HGT, they are one of the machineries implicated in the ability of a bacterium to gain antibiotic resistance. This protocol provides a detailed purification strategy for the Type IV restriction endonuclease SauUSI from Staphylococcus aureus. This protocol eventually leads to ≥95% purity of protein which can then be used for crystallographic and biochemical purposes.

Graphic abstract:

Workflow for purification of SauUSI.

With the recent availability of the SARS-CoV-2 mRNA-based vaccines, public attention has been drawn to this new technology and how it may be applied to other indications. Temporal activation of key hepatic regenerative pathways can induce liver regeneration, overcoming the lack of donor organs for liver transplantation and ineffectiveness of alternative treatments. Recombinant protein therapies and genetic therapies that target these pathways require frequent and repeated injections or, when integrated into the genome, may lead to deleterious effects. In contrast, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) are non-integrative and induce transient yet robust expression of proteins that could serve as an ideal therapeutic tool to treat specific liver diseases. For instance, our recent publication in Nature Communications used mRNA-LNP to express hepatic mitogens, hepatocyte growth factor, and epidermal growth factor to induce liver regeneration following both acute and chronic liver injuries. Initial testing with firefly luciferase mRNA-LNP transfection and in vivo imaging confirmed specific hepatotropic delivery. In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses. This procedure presents an unprecedented tool that can be customized with mRNA-LNP encoding any protein of interest to be expressed by virtually all hepatocytes, ~70% endothelial cells, and ~40% Kupffer cells for promoting liver function and/or regeneration.

Graphic abstract:

Experimental Design of mRNA-LNP IV Injection and Analysis of Liver Cell Specificity and Efficiency of Transfection (Created with BioRender.com)