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.

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.

Protein purification is a critical step in both life sciences and biomanufacturing. Traditional affinity chromatography (AC) methods, including His-tag-based purification, provide high-purity proteins but are limited by the high cost of resins and the need for additional tag-removal steps. In this protocol, we present a reusable SpyDock-modified epoxy resin coupled with a pH-inducible self-cleaving intein for direct purification of proteins with authentic N-termini. This method enables efficient protein purification from cell lysates, achieving high purity (>90%) and yields comparable to the His-tag approach, without requiring tag removal. The SpyDock-modified resin protocol is robust, easy to implement, and cost-effective, making it suitable for both research and large-scale industrial applications.

Antibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A–based affinity chromatography, the gold standard for antibody purification, provides high specificity but can be further improved to increase loading capacity and reduce costs. In this protocol, we introduce a novel approach for purifying high-quality, high-purity antibodies from complex samples using SpyFixer/Z domain–modified resin. This method utilizes Spy chemistry for site-specific immobilization of the Z domain of Protein A, significantly enhancing antibody loading capacity up to 200 mg/mL resin and ensuring stable, durable immobilization. Using this protocol, we achieved >90% purity of human immunoglobulin G (hIgG) from diverse sources, including E. coli cell lysates, human serum, and industrial fermentation broth. The SpyFixer/Z domain–modified resin protocol is simple, cost-effective, and offers a robust, scalable solution for efficient antibody purification in bioengineering applications. This immobilization scheme based on Spy chemistry can also be extended to other immunoglobulin-binding proteins, such as Protein G and Protein L, to develop high-efficiency affinity resins.

The Mediator, a multi-subunit protein complex in all eukaryotes, comprises the core mediator (cMED) and the CDK8 kinase module (CKM). As a molecular bridge between transcription factors (TFs) and RNA polymerase II (Pol II), the Mediator plays a critical role in regulating Pol II–dependent transcription. Considering its large size and complex composition, conducting in vitro studies on the Mediator complex is challenging, especially when isolating the intact and homogeneous complex from human cells. Here, we present a method to purify the intact CKM-cMED complex from FreeStyle 293-F cells (293-F cells), which offers advantages for performing large-scale protein purification. To isolate the CKM-bound cMED without the presence of Pol II, FLAG-tagged CDK8, a subunit of the CKM complex, was expressed in 293-F cells for purification, as CKM and Pol II are mutually exclusive in their interaction with cMED. The complex is isolated from nuclear extracts through immunoaffinity purification and further purified by glycerol gradient to enhance its homogeneity. This protocol provides a time- and cost-efficient way to purify the endogenous Mediator complex for structural- and functional-based studies.

Fatty acid (FA) biosynthesis is a crucial cellular process that converts nutrients into metabolic intermediates necessary for membrane biosynthesis, energy storage, and the production of signaling molecules. Acetyl-CoA carboxylase (ACACA) plays a pivotal catalytic role in both fatty acid synthesis and oxidation. This cytosolic enzyme catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which represents the first and rate-limiting step in de novo fatty acid biosynthesis. In this study, we developed a rapid and effective purification scheme for separating human ACACA without any exogenous affinity tags, providing researchers with a novel method to obtain human ACACA in its native form.

Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology.

MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria Spiroplasma citri, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein.

Chromogranin B and other members of the granin protein family form condensates that recruit clients like proinsulin. The condensation in the lumen of trans-Golgi network (TGN) is critical for the biogenesis of secretory granules. Here, we describe a protocol to purify the tagged version of chromogranin B close to its native form at the TGN, which can then be utilized for microscopy-based assays to monitor condensate formation in vitro and client partitioning depending on the material properties of chromogranin B assemblies.