生物化学

Generating protein conjugates using the bioorthogonal ligation between tetrazines and trans-cyclooctene groups avoids the need to manipulate cysteine amino acids; this ligation is rapid, site-specific, and stoichiometric and allows for labeling of proteins in complex biological environments. Here, we provide a protocol for the expression of conjugation-ready proteins at high yields in Escherichia coli with greater than 95% encoding and labeling fidelity. This protocol focuses on installing the Tet2 tetrazine amino acid using an optimized genetic code expansion (GCE) machinery system, Tet2 pAJE-E7, to direct Tet2 encoding at TAG stop codons in BL21 E. coli strains, enabling reproducible expression of Tet2-proteins that quantitatively react with trans-cyclooctene (TCO) groups within 5 min at room temperature and physiological pH. The use of the BL21 derivative B95(DE3) minimizes premature truncation byproducts caused by incomplete suppression of TAG stop codons, which makes it possible to use more diverse protein construct designs. Here, using a superfolder green fluorescent protein construct as an example protein, we describe in detail a four-day process for encoding Tet2 with yields of ~200 mg per liter of culture. Additionally, a simple and fast diagnostic gel electrophoretic mobility shift assay is described to confirm Tet2-Et encoding and reactivity. Finally, strategies are discussed to adapt the protocol to alternative proteins of interest and optimize expression yields and reactivity for that protein.

Efficient and nontoxic delivery of foreign cargo into cells is a critical step in many biological studies and cell engineering workflows with applications in areas such as biomanufacturing and cell-based therapeutics. However, effective molecular delivery into cells involves optimizing several experimental parameters. In the case of electroporation-based intracellular delivery, there is a need to optimize parameters like pulse voltage, duration, buffer type, and cargo concentration for each unique application. Here, we present the protocol for fabricating and utilizing a high-throughput multi-well localized electroporation device (LEPD) assisted by deep learning–based image analysis to enable rapid optimization of experimental parameters for efficient and nontoxic molecular delivery into cells. The LEPD and the optimization workflow presented herein are relevant to both adherent and suspended cell types and different molecular cargo (DNA, RNA, and proteins). The workflow enables multiplexed combinatorial experiments and can be adapted to cell engineering applications requiring in vitro delivery.

Agrobacterium-mediated transient gene expression in Nicotiana benthamiana is widely used to study gene function in plants. One dramatic phenotype that is frequently screened for is cell death. Here, we present a simplified protocol for Agrobacterium-mediated transient gene expression by infiltration. Compared with current methods, the novel protocol can be done without a centrifuge or spectrometer, thereby suitable for K-12 outreach programs as well as rapidly identifying genes that induce cell death.

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.