Method For Purification Of Proteins Research Articles

Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Stable and reusable calcium-responsive biopolymer for affinity precipitation of therapeutic antibodies.

Affinity precipitation is an attractive method for protein purification due to its many advantages, including the rapid capture of target proteins, simple processing, high specificity, and ease of scale-up. We previously reported a robust antibody purification method using Ca2+-dependent precipitation of ZZ-hCSQ2, a fusion protein of human calsequestrin 2, and the antibody-binding protein ZZ. However, the stability of this fusion protein was not sufficiently high for industrial use because the antibody recovery yield decreased to 60% after being reused 10 times. To identify a more stable calsequestrin (CSQ), we calculated Rosetta energy values for the folding stabilities of various CSQ homologs and selected human CSQ1 (hCSQ1) with lowest energy value (-992.6) as the new CSQ platform. We also identified that the linker sequence between ZZ and CSQ was vulnerable to proteases and alkaline pH by N-terminal protein sequencing. Therefore, we changed the linker to four asparagine (4N) sequences, which were shorter and less flexible than the previous glycine-rich linker. The new version of ZZ-CSQ, ZZ-4N-hCSQ1, was stable in a protease-containing conditioned medium obtained from the cultured Chinese hamster ovary cell or high pH condition (0.1M sodium hydroxide) for more than 5 days and could be reused at least 25 times for antibody purification without loss of recovery yield. The antibodies purified by ZZ-4N-hCSQ1 precipitation also showed greater purity (~33.6-fold lower host cell DNA and ~6.4-fold lower host cell protein) than those purified by protein A chromatography. These data suggest that ZZ-4N-hCSQ1 precipitation is more efficient and can achieve cost-effectiveness of up to 12.5-fold cheaper than previous antibody purification methods and can lower the production costs of therapeutic antibodies.

Separation methods for food protein purification and analysis

The extraction, separation, and purification of dietary proteins from a variety of food sources are crucial for their targeted use in food applications. To achieve this, proteins should be effectively separated from non-protein components such as cell wall structures, polysaccharides, and lipids. Traditional protein purification methods can be time-consuming, highlighting the need for automated, cost-effective, and sustainable alternatives. This comprehensive review critically assesses various protein purification instruments from an analytical perspective, weighing their advantages and disadvantages. The methods under evaluation include ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fast protein liquid chromatography (FPLC), high-performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), and microfluidic chips. Among these, FPLC stands out as an affordable and efficient technique that allows for high protein recovery. However, HPLC and UPLC provide faster results but may denature proteins, leading to lower recovery rates. Ultrafiltration is a cost-effective and straightforward method that doesn’t require complex equipment. Microchip-based approaches are emerging as innovative techniques for rapidly analyzing small samples. While SDS-PAGE is user-friendly, it denatures proteins, particularly those linked to other biomolecules. The choice of the most appropriate instrument depends on factors such as cost, energy efficiency, processing time, the characteristics of the target protein, desired outcomes, protein recovery, and resource availability. By critically examining these analytical instruments for protein purification, this review aims to assist researchers and practitioners in selecting the most suitable method for their specific needs, ultimately promoting efficient and successful protein purification endeavors in the field of food science and technology.

A Brief Review on Separation Techniques: Chromatography Techniques

Chromatography is a system that helps distinct, purify, and recognize mixture components for both quantitative as well as qualitative study [1]. Proteins can be separated and purified depends on their dimensions, shape, net charge, aquaphobic structures on the outermost layer, and binding capability with the immobile phase. The exchange of ions, superficial adsorption, separation, and size exclusion are examples of molecular property and interaction-based separation techniques. Stationary bed chromatography procedures, such as paper chromatography technique, Column Chromatography technique as well as Thin Layer Chromatography technique. Column chromatography technique serves as one of the most common protein purification methods. This review comprises an insight the separation techniques such as Column Chromatography technique, Gas Chromatography technique, Paper Chromatography technique, Thin Layer Chromatography technique, Hydrophobic Interaction Chromatography technique (HIC), used for separation of biomolecules.

Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori.

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.

Construction, heterological expression and a simple purification of the BP region of the pneumococcal surface protein A fused in different orientations to the chemotaxis adaptor protein CheW from Thermotoga petrophila

BackgroundThe important challenge to the biotechnology is to find new effective fusion partners that enable to improve solubility, expression, and optimize the subsequent fine purification of the target protein. ResultsThe most invariant part of the most immunogenic region of the surface virulence factor A of Streptococcus pneumoniae was selected as a model target protein, while the thermostable chemotaxis polypeptide of W-type from Thermotoga petrophila was used as a fusion partner. The genes encoding fusion variants of these proteins were constructed and cloned into a plasmid vector under the control of the strong bacteriophage T7 transcription regulatory system. Effective Escherichia coli producer strains were obtained, and optimal conditions were chosen for the production of resulting constructs. The optimal pH and temperature ranges of recombinant proteins were determined, and three-dimensional shapes of their molecules were also predicted. Methods of low-stage protein purification were improved. Some of the isolated proteins demonstrated a high level of expression, solubility and purity. ConclusionsNovel chimeric proteins were obtained which had not previously been observed in nature in such domain combinations. It was shown that the biotechnologically valuable characteristics of the hybrid proteins were more marked when the thermal-resistant partner was combined with the N-terminus of pneumococcal protein. The principles of their low-stage purification were performed which does not require any special equipment. That is a basis for significant reduction of prices for diagnostic test-systems components and subunit vaccine production.How to cite: Grishin DV, Sidorov NG, Parfenova OK, et al. Construction, heterological expression and a simple purification of the BP region of the pneumococcal surface protein a fused in different orientations to the chemotaxis adaptor protein CheW from Thermotoga petrophila. Electron J Biotechnol 2024;71. https://doi.org/10.1016/j.ejbt.2024.05.001.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Phase separation methods for protein purification: A meta-analysis of purification performance and cost-effectiveness.

Protein purifications based on phase separations (e.g., precipitation and liquid-liquid extraction) have seen little adoption in commercial protein drug production. To identify barriers, we analyzed the purification performance and economics of 290 phase separation purifications from 168 publications. First, we found that studies using Design of Experiments for optimization achieved significantly greater mean yield and host cell protein log10 removal values than those optimizing one factor at a time (11.5% and 53% increases, respectively). Second, by modeling each reported purification at scales from 10 to 10,000kg product/year and comparing its cost-effectiveness versus chromatography, we found that cost-effectiveness depends strongly on scale: the fraction of phase separations predicted to be cost-effective at the 10, 100, and 1000kg/year scales was 8%, 15%, and 43%, respectively. Total cost per unit product depends inversely on input purity, with phase separation being cheaper than chromatography at the 100kg/year scale in 100% of cases where input purity was ≤ 1%, compared to about 25% of cases in the dataset as a whole. Finally, we identified a simple factor that strongly predicts phase separation process costs: the mass ratio of reagents versus purified product (the "direct materials usage rate"), which explains up to 58% of variation in cost per unit of purified product among all 290 reports, and up to 98% of variation within particular types of phase separation.

A Novel 3D-Printed and Miniaturized Periodic Counter Current Chromatography System for Continuous Purification of Monoclonal Antibodies.

Continuous chromatography has emerged as one of the most attractive methods for protein purification. Establishing such systems involves installing several chromatographic units in series to enable continuous separation processes and reduce the cost of the production of expensive proteins and biopharmaceuticals (such as monoclonal antibodies). However, most of the established systems are bulky and plagued by high dead volume, which requires further optimization for improved separation procedures. In this article, we present a miniaturized periodic counter-current chromatography (PCCC) system, which is characterized by substantially reduced dead volume when compared to traditional chromatography setups. The PCCC device was fabricated by 3D printing, allowing for flexible design adjustments and rapid prototyping, and has great potential to be used for the screening of optimized chromatography conditions and protocols. The functionality of the 3D-printed device was demonstrated with respect to the capture and polishing steps during a monoclonal antibody purification process. Furthermore, this novel miniaturized system was successfully used for two different chromatography techniques (affinity and ion-exchange chromatography) and two different types of chromatographic units (columns and membrane adsorbers). This demonstrated versability underscores the flexibility of this kind of system and its potential for utilization in various chromatography applications, such as direct product capture from perfusion cell cultures.

Capture of endogenous lipids in peptidiscs and effect on protein stability and activity

Compared to protein-protein and protein-nucleic acid interactions, our knowledge of protein-lipid interactions remains limited. This is primarily due to the inherent insolubility of membrane proteins (MPs) in aqueous solution. The traditional use of detergents to overcome the solubility barrier destabilizes MPs and strips away certain lipids that are increasingly recognized as crucial for protein function. Recently, membrane mimetics have been developed to circumvent the limitations. In this study, using the peptidisc, we find that MPs in different lipid states can be isolated based on protein purification and reconstitution methods, leading to observable effects on MP activity and stability. Peptidisc also enables re-incorporating specific lipids to fine-tune the protein microenvironment and assess the impact on downstream protein associations. This study offers a first look at the illusive protein-lipid interaction specificity, laying the path for a systematic evaluation of lipid identity and contributions to membrane protein function.

One-Step Purification of Recombinant Cutinase from an E. coli Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand.

Cutinase from Fusarium solani pisi is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from E. coil extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3', [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant Ka ≈ 104 M-1) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an E. coli crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%.

Protein Isolation from 3D Hydrogel Scaffolds.

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.

GCLiPP: global crosslinking and protein purification method for constructing high-resolution occupancy maps for RNA binding proteins

GCLiPP is a global RNA interactome capture method that detects RNA-binding protein (RBP) occupancy transcriptome-wide. GCLiPP maps RBP-occupied sites at a higher resolution than phase separation-based techniques. GCLiPP sequence tags correspond with known RBP binding sites and are enriched for sites detected by RBP-specific crosslinking immunoprecipitation (CLIP) for abundant cytosolic RBPs. Comparison of human Jurkat T cells and mouse primary T cells uncovers shared peaks of GCLiPP signal across homologous regions of human and mouse 3′ UTRs, including a conserved mRNA-destabilizing cis-regulatory element. GCLiPP signal overlapping with immune-related SNPs uncovers stabilizing cis-regulatory regions in CD5, STAT6, and IKZF1.

Regulation of BCMA By the Ubiquitin Proteasome System Enables Optimization of BCMA-Targeting Therapies in Multiple Myeloma

Background: The exclusive expression of the B-cell maturation antigen (BCMA), in normal and malignant plasma cells, currently makes it a prominent target for immune cell therapies in multiple myeloma (MM) including chimeric antigen receptor T-cell (CART) and bi-specific T cell engaging antibody (BiTE) therapies. Despite initial high response rates, most patients eventually relapse highlighting the need to enhance therapy efficacy. Although loss of BCMA-antigen expression marks one reason for therapy failure, little is known about the mechanistic means of antigen escape. The ubiquitin proteasome system (UPS) controls abundance and protein degradation of about 80-90% of all cellular proteins. Protein degradation is initiated by substrate ubiquitination, which is orchestrated by the sequential action of enzymes whereby only E3-ligases possesses substrate specificity. Deubiquitinating enzymes (DUBs) can reverse substrate ubiquitination. Against this background, we raised the question whether BCMA antigen presentation, expression, and activity is regulated by the UPS and whether the UPS can be utilized to enhance BCMA directed CART therapies in MM. Methods: Protein turnover and regulation by the UPS was assayed using cycloheximide and proteasomal inhibitors (PI) followed by immunoblotting. Direct substrate ubiquitylation was confirmed by ubiquitylation assays based on immunoprecipitation and image-based analysis. Changes in antigen surface presentation were determined using FACS analysis. Changes in cellular signaling and downstream effects were assessed by immunoblotting and quantitative PCR (qPCR). Primary CD138 + cells were obtained from bone marrow aspirations followed by MACS ® cell separation. To assess the effect of PI on CART killing capacity, a classic co-culture was set up in vitro. Using a proximity-based protein purification method (TurboID), UPS-related interactors of BCMA were identified. Results: For the first time, we show that BCMA is a short-lived protein with a half-life of approximately 30 min in MM cells. Degradation of BCMA is abrogated upon PI treatment. Inhibition of γ-secretase, an established protease of the extracellular domain of BCMA does not impair BCMA turnover. Furthermore, we show that BCMA is indeed ubiquitylated in MM cells and that ubiquitylation is induced by the ligand APRIL. These results support the hypothesis that the UPS regulates BCMA abundance. In both MM cell line models and primary CD138 + MM cells of PI refractory/ relapsed MM (RRMM) patients, inhibition of the proteasome leads to a significant increase of BCMA on the cell surface without promoting oncogenic NF-κB signaling in MM cells. Subsequently, we show that the increase in BCMA surface expression upon PI treatment substantially increases the efficacy of BCMA directed CART cells in co-cultures with MM cells and primary CD138 + MM patient cells. Specific members of the UPS (E3 ligases and DUBs) identified from our proteome wide BCMA interactome analysis are currently being validated. Conclusion: Here we show, for the first time, that BCMA is ubiquitylated and degraded by the ubiquitin proteasome system in multiple myeloma. The increase in BCMA expression upon proteasomal inhibition enhanced efficacy of BCMA-targeting CART cells. These insights provide a rationale for combining BCMA CART cells with PI in patients with BCMA CART relapsed/refractory disease and persistence. The identification of BCMA-specific E3-ligases and DUBs may represent novel therapeutic targets to specifically prevent BCMA degradation and antigen escape.

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase.

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA-protein complexes or PEI-acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme's properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion.

Immunoaffinity Chromatography for Protein Purification and Analysis.

Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low-performance supports such as agarose for off-line immunoextraction and supports used in high-performance IAC for on-line immunoextraction. The use of IAC for protein analysis as a flow-based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Off-line immunoextraction by traditional immunoaffinity chromatography Basic Protocol 2: On-line immunoextraction by high-performance immunoaffinity chromatography Basic Protocol 3: Competitive binding chromatographic immunoassay.

Soluble expression and purification of recombinant bovine ferritin H-chain

Ferritin is a potential medicine delivery vehicle and vaccine platform, and its efficient expression is a prerequisite for widespread application. This study introduces a soluble expression strategy for recombinant bovine ferritin heavy chain (rFTH) in a prokaryotic system and an improved protein purification method. The amplified rFTH gene was ligated into the prokaryotic expression vector pET30a. The recombinant vectors with the N-terminal His-tag(N-His) or C-terminal His-tag(C-His) were translated and expressed separately. The results showed that the solubility of rFTH with C-His was significantly higher than that with N-His. The expression of rFTH with C-His was attempted at 37 °C and 16 °C, respectively. The results showed that the proportion of soluble protein expressed at 37 °C was more than 90%, higher than that expressed at 16 °C. Then rFTH with C-His was purified successfully using anion exchange chromatography, modified PEG precipitation, and dialysis. The rFTH protein was characterized using SDS-PAGE, Native-PAGE, Western blot, transmission electron microscopy, and dynamic light scattering. The results demonstrated that the purified rFTH protein self-assembled into ferritin nanoparticles with a regular shape and uniform size. This study sheds new light on the soluble expression of ferritin and provides a foundation for the construction of bovine ferritin nanoparticle production platforms.

Chromatographic purification of histidine-tagged proteins using zirconia particles modified with phosphate groups

Immobilized metal ion affinity chromatography (IMAC) is one of the most common purification techniques for histidine-tagged proteins (His-tagged proteins). IMAC enables the purification of His-tagged proteins at high purity on the basis of coordination bonds between His-tags and metal ions (such as Ni2+, Co2+, and Cu2+) immobilized on the matrices in columns. However, IMAC requires low-pH solutions or high-concentration imidazole solutions for eluting His-tagged proteins, which can affect protein conformation and activity. The present study provides a His-tagged protein purification method using zirconia particles modified with phosphate groups. This method is based on the electrostatic attractions between a His-tag moiety of proteins and phosphate groups on the zirconia particles; this method requires only high-concentration salt solutions at pH 7.0 for eluting the proteins. A column packed with phosphate-modified zirconia particles was demonstrated to enable the purification of two model His-tagged proteins—His-tagged green fluorescent protein and His-tagged alkaline phosphatase fused with maltose binding protein. Thus, this chromatography method is useful for purifying His-tagged proteins without any pH stress or additives. Additionally, because of the mechanical properties of the zirconia particles, this technique enables high-performance purification at a high flow rate.

Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different E. coli strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

Analysis of histidine-tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry.

Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). A protein standard, His-Ubq, and two recombinant proteins, His-SHAN and His-CS, expressed in Escherichia coli were immobilized on two immobilized metal affinity systems, Cu-nitriloacetic acid (Cu-NTA) and Ni-NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96-well plate form factor, or analyzed directly from immobilized metal affinity-coated microscope slides by DESI-MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. Small proteins (His-Ubq) and medium proteins (His-SAHN) could readily be detected from 96-well plates by direct infusion ESI, or from microscope slides by DESI-MS after purification on surface from clarified E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu-NTA and Ni-NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His-SAHN and the methylation product of His-CS (theobromine to caffeine) were detected. The immobilization, purification, release and detection of His-tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI-MS or ambient DESI-MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.