生物化学

Protein purification is essential for drug development, antibody production, and structural biology. We propose a cost-effective chromatography method using elastin-like polypeptide (ELP) as an aggregating core. In this approach, a chilled (target protein)-ELP fusion is loaded onto an immobilized metal affinity chromatography (IMAC) column equilibrated with low-salt buffer. Impurities are removed with warm high-salt buffer washes. Warming the column above the ELP’s transition temperature (T_m) triggers ELP aggregation, physically trapping the target protein between beads. Subsequent stringent washing (high salt/imidazole) eliminates residual contaminants. Finally, cooling with cold low-salt buffer reverses aggregation, eluting the purified target protein. This method eliminates the need for advanced chromatography systems while achieving high purity through dual mechanisms: (1) IMAC affinity and (2) temperature-dependent physical capture. The ELP’s reversible phase transition offers a simplified yet efficient purification platform, particularly valuable for lab-scale production of challenging proteins.

The voltage-gated proton channel (H_v1) is a membrane protein that dissipates acute cell proton accumulations. To understand the molecular mechanisms explaining H_v1 function, methods for purifying the protein are needed. Previously, methods were developed for expressing and purifying human H_v1 (hH_v1) in yeast and later in bacteria. However, these methodologies produced low protein yields and had high production costs, considerably limiting their usefulness. The protocol described in this work was developed to overcome those limitations. hH_v1 is overexpressed in bacteria, solubilized with the detergent Anzergent 3–12, and purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Our protocol produced higher protein yields at lower costs than previously published methodologies.

The study of carbohydrate–protein interactions is crucial for clarifying biological processes and identifying potential drug candidates. However, due to the complex structure of carbohydrates, such as high molecular weight, dynamic flexibility, and high solution viscosity, it is challenging to study their interactions with diverse proteins. Conventional analytical techniques like isothermal titration calorimetry (ITC), X-ray crystallography, molecular dynamics (MD) simulations, and nuclear magnetic resonance (NMR) spectroscopy have limitations in revealing these molecular interactions. Surface plasmon resonance (SPR), an advanced optical biosensor technique, overcomes these limitations. It enables real-time, label-free monitoring of the interaction dynamics between carbohydrates and proteins through a continuous flow over a chip surface. In this study, we utilized SPR-based techniques to explore the interaction of capsular polysaccharides (CPS) of Klebsiella pneumoniae and the enzyme KpACE (K. pneumoniae acetylated capsule esterase). Our SPR-based analytical platform has several advantages, including shorter experimental time, a simulated physiological state, and minimal sample requirements for investigating carbohydrate–protein interactions. This approach expands the applicability scope of SPR technology and provides a valuable tool for a wide range of research. By using SPR, we successfully verified that KpACE acts on the acetyl groups of CPS, demonstrating its enzymatic activity, which is crucial for understanding the pathogenic mechanism of K. pneumoniae and developing potential antibacterial drugs.

Intermediate states are often populated during the folding and unfolding reactions of a protein, and their detection is very challenging as they form transiently. Structural characterization of these short-lived intermediate species is difficult as it requires high-resolution methodologies. Hydrogen exchange-mass spectrometry (HX-MS) can identify and yield direct structural information on folding and unfolding intermediates, as well as information about the cooperativity of the folding or unfolding processes. The mass distributions of intact protein molecules are obtained first to determine their exchange pattern. Then, segment-specific structural information is obtained by analyzing the fragments of the protein. Enzymatic digestion is widely used with HX to determine the sequence-specific structural changes that occur to the protein during folding or unfolding. However, if a protein is an inhibitor of the protease, then alternative methodologies are required. Using electron transfer dissociation (ETD), it is possible to fragment the protein inside a mass spectrometer, and segment-specific structural changes occurring during the folding and unfolding process can be determined. In the case of HX-ETD-MS, protein molecules are first allowed to undergo HX, followed by their fragmentation. Deuterium retention in each fragment is measured. Very little, if any, scrambling of deuterium across fragments occurs during ETD-enabled fragmentation; hence, there is little scope for misinterpretation of the HX data.

Ubiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual function of 1) transferring covalently attached ubiquitin from the catalytic cysteine of E2 (E2~Ub) to the substrate and 2) providing substrate specificity. Our current knowledge of their individual substrate pools is incomplete due to the difficult capture of these transient substrate–E3 ligase interactions. Here, we present an efficient protocol that enables the selective biotinylation of substrates of a given ubiquitin ligase. In brief, the candidate E3 ligase is fused to the biotin ligase BirA and ubiquitin to a biotin acceptor peptide, an Avi-tag variant (-2) AP. Cells are co-transfected with these fusion constructs and exposed to biotin, resulting in a BirA-E3 ligase-catalyzed biotinylation of (-2) AP-Ub when in complex with E2. As the next step, the biotinylated (-2) AP-Ub is transferred covalently to the substrate lysine, which enables an enrichment via denaturing streptavidin pulldown. Substrate candidates can then be identified via mass spectrometry (MS). Our ubiquitin-specific proximity-dependent labeling (Ub-POD) method allows robust biotinylation of the ubiquitylation substrates of a candidate E3 ligase thanks to the wild-type BirA and biotin acceptor peptide fused to the E3 and Ub, respectively. Because of the highly Ub-specific labeling, Ub-POD is more appropriate for identifying ubiquitination substrates compared to other conventional proximity labeling or immunoprecipitation (IP) approaches.

X-succinate synthase enzymes (XSSs) are a class of glycyl radical enzymes (GREs) that play a pivotal role in microbial anaerobic hydrocarbon degradation. They catalyze the addition of hydrocarbons to fumarate using a protein-based glycyl radical, which must first be installed by a radical S-adenosylmethionine (rSAM) activating enzyme (AE). Once activated, XSS enzymes can undergo multiple catalytic cycles, forming C(sp³)–C(sp³) bonds with high stereoselectivity—a feature that highlights their potential as asymmetric biocatalysts. Due to the insolubility of XSS-AEs when heterologously expressed in Escherichia coli, studies have relied on in vivo radical installation protocols. Although these methods have illuminated fundamental details of XSS mechanisms, the inability to install a glycyl radical in vitro has limited biochemical studies and biotechnological advances using these enzymes. Here, we describe an in vitro protocol for reconstituting the activity of benzylsuccinate synthase (BSS), an XSS that catalyzes the addition of toluene to fumarate to form R-benzylsuccinate. To enable in vitro glycyl radical installation, we identified a soluble homolog via genome mining: 4-isopropylbenzylsuccinate synthase activating enzyme (IbsAE). IbsAE was expressed in E. coli and anaerobically purified in moderate yields (6–8 mg of protein per liter of culture); herein, we outline the expression and anaerobic purification of both IbsAE and BSS proteins. We describe a reproducible method for in vitro glycyl radical installation using these recombinant proteins and provide guidance on quantifying radical formation. Our optimized protocol consistently achieves 30%–50% radical installation, comparable to other in vitro GRE activations. Lastly, we demonstrate the application of this protocol for in vitro hydroalkylation reactions, achieving high assay yields (89%–97%). This protocol enables biochemical experiments that were previously challenging using cell extracts and accelerated advancements in XSS engineering and use in biocatalysis.

N⁶-methyladenosine (m6A) is an abundant internal mRNA modification with roles in regulating cellular and organismal physiology, including development, differentiation, and disease. The deposition of m6A is highly regulated, with various m6A levels across different environmental conditions, cellular states, and cell types. Available methods for measuring bulk m6A levels are often time-consuming, have low throughput, and/or require specialized instrumentation or data analyses. Here, we present a detailed protocol for measuring bulk m6A levels in purified poly(A) RNA samples with m6A-ELISA using a standard-based approach. Critical steps of the protocol are highlighted and optimized, including poly(A) RNA quality controls and antibody specificity testing. The protocol is fast, scalable, adaptable, and cost-effective. It does not require specialized instrumentation, training, or skills in data analysis. We have successfully tested this protocol on mRNAs isolated from budding yeast and mouse cell lines.

De novo synthesis of purine nucleotide is essential for the production of genetic materials and cellular chemical energy. PRPP amidotransferase (PPAT) is the rate-limiting enzyme in de novo purine synthesis, thereby playing a crucial regulatory role in this pathway. Recent studies suggest that metabolic enzymes, including PPAT, form condensates through phase separation to regulate cellular metabolism in response to environmental changes. However, due to the lack of methods for purifying eukaryotic PPAT, the biophysical properties of the enzyme have remained unknown. Here, I describe a protocol for purifying budding yeast PPAT tagged with green fluorescent protein from yeast cells, as well as an in vitro assay to examine condensation of the fluorescent PPAT by microscopy. These techniques enabled us to elucidate the mechanism controlling PPAT condensation and may also be applicable to the purification and condensation assay of other enzymes.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane, where it plays important roles in membrane trafficking and cytoskeletal dynamics through proteins that directly bind to it. PI(4,5)P₂ can be metabolized to other phosphorylated forms of phosphatidylinositol to regulate numerous processes such as cell growth and development. PI(4,5)P₂ can also be hydrolyzed to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP₃). Altered metabolism or mislocalization of PI(4,5)P₂ can perturb one or more of its functions and contribute to disease states. Here, we present a protocol to visualize and quantify the localization of PI(4,5)P₂ in live cells. The protocol uses a highly specific PI(4,5)P₂ protein binding domain coupled to enhanced green fluorescence protein (PH-PLCD1-GFP), enabling localization and quantification of cytosol-facing PI(4,5)P₂ to be determined. Localization and quantification of the PH-PLCD1-GFP, PI(4,5)P₂ specific probe, is enabled by fluorescence imaging and confocal microscopy. This approach can be used to study the dynamics of PI(4,5)P₂ localization temporally in live cells under both physiological and pathological conditions.

We have observed that some proinsulin molecules in pancreatic islets and beta cell lines have incomplete or improper intramolecular disulfide bonds. These misfolded monomers can form intermolecular disulfide-linked complexes. Accurately quantifying the fraction of proinsulin molecules contained in these complexes is challenging. By proinsulin immunoblotting after nonreducing SDS-PAGE, the signal for disulfide-linked complexes can exceed the total proinsulin signal detected after reducing SDS-PAGE (i.e., overestimating the abundance of misfolded species due to antibody affinity differences). However, after modification of the SDS-PAGE and electrotransfer protocol, we have been able to more accurately estimate the fraction of proinsulin monomers folded to the native state, as well as misfolded proinsulin monomers and disulfide-linked complexes. Our improved technique offers the ability to obtain a more precise assessment of proinsulin misfolding under different environmental conditions in beta cells and normal islets and in diabetes.