Purification Conditions Research Articles

Data Mining for the Characterization of a Paper Prototype Obtained with Bacterial Cellulose Derived from Banana and Pineapple By-Products

The primary objective of this research is to evaluate the feasibility of two of the most prevalent agricultural residues in Ecuador, banana peels and pineapple peels, as a carbon source in the culture medium of Komagataeibacter hansenii for the production of bacterial cellulose (BC) and BC-based paper. This analysis includes an assessment of the productivity parameters of the obtained BC and the quality parameters of the BC-based paper, employing multivariate statistical methodologies. The experimental design consisted of fifteen treatments: T1 served as the control using the standard HS medium, while treatments T2–T8 used banana peel extracts (BPE), and treatments T9–T15 used pineapple peel extracts (PPE) at concentrations from 10% to 40% (v/v). Extracts were prepared with tailored pretreatments for each type of peel to optimize bioactive compound recovery. Standardized fermentation and purification conditions were applied, and once the cellulose was obtained, additives and coating agents were incorporated to produce paper samples from each treatment. The results indicated that higher BPE concentrations (T5, T6, T7, and T8) correlated significantly with increased Weight and Yield of BC, as well as improved grammage and water content in the BC-based paper. This highlights that efficient paper production is influenced by the quality of the bacterial cellulose used, with BPE-based media yielding optimal results due to their nutrient composition, which promotes bacterial growth and metabolic activity. This approach suggests a pathway for advancing sustainable and economical paper production.

Scaling down recombinant carbonic anhydrase isolation with immobilized metal ion chromatography (IMAC): Harnessing enzymatic carbon dioxide capture and mineralization

BackgroundHuman activities have led to increased atmospheric CO2 levels, raising concerns about climate change. Carbonic anhydrase (CA) enzymes show promise for transforming CO2 into valuable products like calcium carbonate (CaCO3) through mineralization. Purifying and immobilizing CA enzymes on nanofiber membranes enhances their catalytic activity, enabling efficient CO2 conversion and mineralization. MethodsRecombinant CA was purified using immobilized metal affinity chromatography (IMAC), optimizing pH, biomass concentration, flow rate, and loading volume for maximum efficiency. The CA enzyme was then immobilized onto a weak ion exchange nanofiber membrane functionalized with AEA-COOH to create the CA-modified membrane (AEA-COOH-CA), enhancing CO2 conversion and CaCO3 mineralization. Significant findingsOptimal purification conditions (pH 7, 1 % biomass, 0.1 mL/min flow rate, 1.0 mL loading volume) were determined using IMAC. The CA-modified membrane effectively converted CO2 and mineralized CaCO3, demonstrating the potential for environmental CO2 sequestration. The immobilized CA activities of the AEA-COOH-CA nanofiber membranes exhibited 473.42 WAU/g-membrane, corresponding to 7.10 WAU per membrane piece. The CaCO3 precipitation reached 83.90 mg, with a precipitation efficiency of 11.82 mg CaCO3/WAU. These findings underscore the promise of enzymatic carbon capture using CA-modified membranes, offering a sustainable solution for greenhouse gas mitigation.

Efficient Development of Nanobody-Based Affinity Chromatography for AAV8 Purification

Adeno-associated virus serotype 8 (AAV8) is a highly effective vector for gene therapy. However, its purification remains challenging due to its low natural abundance and stringent purity requirements. This study aimed to develop an affinity chromatography resin utilizing nanobodies (Nbs) to enhance AAV8 purification efficiency. An AAV8-specific Nb library was constructed, leading to the identification of Nb9 as the most promising candidate based on its high binding affinity, stability and yield. Nb9 was expressed in Pichia pastoris, resulting in high yield and exceptional purity. Two types of agarose resins, Epoxy activated Bestarose 6B and PabPur SulfoLink Beads 4FF, were employed for Nb9 conjugation. Epoxy activated Bestarose 6B resin exhibited a significantly higher ligand density (9.12 mg/mL). Binding capacity assessments of the LQ01 resin demonstrated optimal performance at pH 7.0, with diminishing efficacy at lower and higher pH levels. Different NaCl concentrations influenced the binding efficiency, providing critical insights for refining purification conditions. Purification trials exhibited high specificity, purity and consistent VP protein ratio, as evidenced by SDS-PAGE analysis, confirming effective AAV8 capture and elution. Furthermore, the resin demonstrated robust performance across repeated cycles, retaining 71.9% of its initial binding capacity after 20 uses and maintaining stability with only a 6% reduction after 7 days at 37°C. These findings highlight LQ01's potential for scalable and cost-effective AAV8 purification, while demonstrating the broader applicability of Nbs in affinity chromatography and biotechnological processes.

Dummy-template imprinted bovine serum albumin for extraction of zearalenone.

The aim of this study is to develop molecularly imprinted protein specific to zearalenone(ZEN). The primary idea of our study was to replace the toxic template-ZEN-with a dummy-template-4-hydroxicoumarin-during the synthesis of imprinted proteins(IPs). The choice of the dummy-template was based on the results of comprehensive evaluation that included a combination of blind docking and molecular dynamics simulations. Furthermore, we studied the influence of protonation and purification conditions to IPssorption capacity. 3Dfluorescence spectroscopy was used to monitor the process of bovine serum albumin(BSA) imprinting. The modified purification approach allowed reducing the eluent volume and purification time by approximately 300 and 48times, respectively. The imprinted BSA was then used to produce a bioinorganic sorbent(IPs-BIS) based on silica nanoparticles (silicaNPs), that, as far as we know, was never described before. The synthesized IPs-BIS were successfully applied as ZEN sorbents in model solutions (Q = 1.70 ± 0.15mg g , imprinting factor = 2.5) and artificially contaminated wheat extract (Q = 2.24 ± 0.02mg g ) confirmed by HPLC-UV. We believe that our method can be used for mycotoxin monitoring in animal feeds and foodstuff.

Green Synthesis of 2-Mercapto 5,6-Dihydro-4H-1,3-Thiazines via Sequential C-S Couplings.

The six-membered N,S-heterocyclic 1,3-thiazines and their derivatives are widely acknowledged as pharmaceutical molecules with a wide range of biological activities. In this study, we developed a unique thiol-involved cascade reaction that enables the efficient construction of the 5,6-dihydro-4H-1,3-thiazine scaffold through consecutive intermolecular thiol-isothiocyanate and intramolecular thiol-halogen click reactions. Structurally diverse 2-mercapto dihydrothiazines including three antitumour candidates of bis-dihydrothiazines were readily obtained in high yields from the readily available thiols and 3-chloroisothiocyanate in the green solvent EtOH/H2O (1:1) using K2CO3 (0.6 equiv.) as the base. Between the two synthesis procedures investigated, the microwave-assisted reaction generally behaved more efficiently than that under routine heating conditions. Furthermore, DFT calculation confirmed the sequential addition-substitution mechanism. This cascade C-S coupling reaction methodology offers several advantages, including rapid completion, high reliability, easy purification, and benign conditions.

Improving Stability of Spiroplasma citri MreB5 Through Purification Optimization and Structural Insights.

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. Key features • The protocol is a useful approach to standardize purification of nucleotide-dependent cytoskeletal filaments and other nucleotide-binding proteins. • The mechanistic basis of how different ions could stabilize a protein, and hence improve yield in purification, has been demonstrated. • The change in buffer conditions/salt enabled us to get sufficient yield for biochemical and structural characterization.

Purification and Characterization of β-Mannanase Derived from Rhizopus microsporus var. rhizopodiformis Expressed in Komagataella phaffii.

The demand for food-grade β-mannanases, ideal for high-temperature baking, is increasing. Using the Komagataella phaffii (P. pastoris) expression system for β-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of β-mannanase (PpRmMan134A) from recombinant P. pastoris X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant P. pastoris in 70 mL buffered glycerol complex medium for 24 h at 30 °C, then in modified buffered methanol-complex medium with 0.91% (w/v) methanol, 0.56% (w/v) sorbitol, and 0.48% (w/v) mannitol for another 24 h, which improved the PpRmMan134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. PpRmMan134A showed thermostability up to 100 °C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified PpRmMan134A emerges as a promising enzyme for high-temperature baking applications.

A 3-step load-wash-elute solid-phase extraction tandem dedicated clean-up cartridge prior to UPLC-MS/MS for the simultaneous determination of bongkretic acid, isobongkretic acid and toxoflavin in edible mushrooms and rice noodles

A 3-step load-wash-elute solid-phase extraction tandem dedicated clean-up cartridge prior to UPLC-MS/MS for the simultaneous determination of bongkretic acid, isobongkretic acid and toxoflavin in food is presented in this work. The 3-step load-wash-elute SPE strategy with PRiME HLB, eliminating conditioning and equilibration, and coupled with dedicated purification cartridge, PriboFast MFC 336, was used as a tandem cartridge for sample pretreatment. Various sample pretreatment conditions including sample extractant, the tandem cartridge, the optimizatioin of PRiME HLB SPE conditions and PriboFast MFC 336 purification conditions, as well as MS/MS conditions and HPLC conditions were systematically investigated. Under the optimal experimental conditions, high sensitivity (LODs: 0.030–0.040 μg kg−1, LOQs: 0.090–0.12 μg kg−1), acceptable recoveries (83 %–96 %) and good precisions (r2 < 6.0 %) were obtained. The method was successfully applied to 90 food samples, bongkretic acid and isobongkretic acid were detected simultaneously in a dried black fungus, whereas toxoflavin was not detected in any of the samples.

Preparation and characterization of insoluble β-glucan from waste beer yeast

The management of brewery waste, particularly waste brewer's yeast, has posed a significant environmental and economic challenge. This study proposed an approach to valorize waste brewer's yeast by extracting β-glucan. The key phases of the research included yeast autolysis, alkaline treatment and optimization of the β-glucan purification conditions. The findings revealed that a 0.7 % NaOH concentration at 80 °C for 2.6 h was the most effective for impurity removal. In the comparative analysis, it was confirmed that extraction at 80 % DMSO concentration with a 30:1 solid-liquid ratio at 80 °C for 30 min was the optimal condition for obtaining high-purity β-glucan. The process yielded a β-glucan high purity of 95.84 ± 1.15 % and a yield of 5.56 ± 0.34 % based on the wet weight after centrifugation. The structure was analyzed using TLC, FTIR and NMR. The extracts are polysaccharides composed of glucose monomers linked by β-glycosidic bonds. In addition, It has demonstrated significant anti-inflammatory properties in vitro and can be developed as a functional food. This study provides a feasible and efficient solution for the sustainable utilization of waste brewer's yeast, contributing to environmental sustainability and offering economic benefits for the brewing industry.

Antidiabetic and Antigout Properties of the Ultrasound-Assisted Extraction of Total Biflavonoids from Selaginella doederleinii Revealed by In Vitro and In Silico Studies.

In this study, the extraction, purification and metabolic enzyme inhibition potential of Selaginella doederleinii were investigated. In order to extract the total biflavonoids from S. doederleinii (SDTBs), the optimum extraction process was obtained by optimizing the ultrasonic extraction parameters using response-surface methodology. This resulted in a total biflavonoid content of 22.26 ± 0.35 mg/g. Purification of the S. doederleinii extract was carried out using octadecylsilane (ODS), and the transfer rate of the SDTBs was 82.12 ± 3.48% under the optimum purification conditions. We determined the effect of the SDTBs on α-glucosidase (AG), α-amylase and xanthine oxidase (XOD) and found that the SDTBs had an extremely potent inhibitory effect on AG, with an IC50 value of 57.46 μg/mL, which was much lower than that of the positive control. Meanwhile, they also showed significant inhibition of XOD and α-amylase, with IC50 values of 289.67 μg/mL and 50.85 μg/mL, respectively. In addition, molecular docking studies were carried out to understand the nature of the action of the biflavonoids on AG and XOD. The results showed that robustaflavone had the lowest binding energy to AG (-11.33 kcal/mol) and XOD (-10.21 kcal/mol), while, on the other hand, amentoflavone showed a good binding affinity to AG (-10.40 kcal/mol) and XOD (-9.962 kcal/mol). Moreover, molecular dynamics simulations verified the above results.

Effect of Freeze-Thawing Treatment on Platelet-Rich Plasma Purified with Different Kits.

Osteoarthritis of the knee (OAK), a progressive degenerative disease affecting quality of life, is characterized by cartilage degeneration, synovial inflammation, and osteophyte formation causing pain and disability. Platelet-rich plasma (PRP) is an autologous blood product effective in reducing OAK-associated pain. PRP compositions depend on their purification. In clinical practice, PRP is typically administered immediately after purification, while cryopreserved PRP is used in research. Platelets are activated by freezing followed by release of their humoral factors. Therefore, PRP without any manipulation after purification (utPRP) and freeze-thawed PRP (fPRP) may differ in their properties. We purified leukocyte-poor PRP (LPPRP) and autologous protein solution (APS) to compare the properties of utPRPs and fPRPs and their effects on OAK target cells. We found significant differences in platelet activation and humoral factor content between utPRPs and fPRPs in both LPPRP and APS. Freeze-thawing affected the anti-inflammatory properties of LPPRP and APS in chondrocytes and synovial cells differed. Both utPRPs and fPRPs inhibited polarization toward M1 macrophages while promoting polarization toward M2 macrophages. Freeze-thawing specifically affected humoral factor production in macrophages, suggesting that evaluating the efficacy of PRPs requires considering PRP purification methods, properties, and conditions. Understanding these variations may enhance therapeutic application of PRPs in OAK.

PRODUCTION, PURIFICATION AND DETERMINATION MOLECULAR WEIGHT OF COAGULASE ENZYME FROM STAPHYLOCOCCUS AUREUS ISOLATED FROM CLINICAL SAMPLES

Background: Coagulase is a critical enzyme produced by pathogenic strains of Staphylococcus aureus, which plays a significant role in the classification of staphylococci into coagulase-positive (CoP) and coagulase-negative staphylococci (CoNS). This enzyme interacts with prothrombin to induce blood clotting, which is essential for understanding its pathogenic mechanisms. Knowledge Gap: Despite its importance, detailed methods for extracting, purifying, and characterizing coagulase from clinical S. aureus isolates remain underexplored, particularly in relation to optimizing purification conditions and accurately determining its molecular weight. Aims: This study aimed to extract and purify coagulase from Staphylococcus aureus isolated from clinical samples and to determine the enzyme's molecular weight using SDS-PAGE. Methods: A total of 2,000 clinical samples were collected from hospitals in Baghdad, yielding 130 isolates of S. aureus. The optimum conditions for coagulase production were identified as pH 7.5 and 37°C. Coagulase was extracted and purified through ammonium sulfate precipitation (50-80% saturation), SDS-PAGE, ion exchange chromatography with DEAE cellulose, and gel filtration using Sephadex G150. Results: The crude coagulase extract exhibited an activity of 1.7 U/ml. Following purification, the enzyme's specific activity was measured, and the molecular weight of the coagulase was determined to be 36 kilodaltons (kDa). Novelty and Implications: This study provides a detailed protocol for the extraction and purification of coagulase from clinical isolates of S. aureus, along with the molecular weight determination of the enzyme. The findings enhance the understanding of coagulase's biochemical properties and its role in staphylococcal pathogenicity, potentially contributing to improved diagnostic and therapeutic strategies.

Optimization of impurity removal and purification flux components and their application in the production of A356 aluminum alloy

Abstract Taking into account the thermodynamic and kinetic conditions of impurity removal and purification, three impurity removal and purification fluxes were designed for comparative experiments. It was found that the impurity and hydrogen removal rates of experimental flux II were the best, with a decrease of 28.1% in micro grain size 28.1 μ m. The spacing between secondary dendrites is significantly reduced. The eutectic silicon phase of the alloy treated with experimental flux II is granular, with small and dispersed inclusions. The fracture structure of the alloy exhibits ductile fracture characteristics, mainly in the form of transgranular microporous aggregation, which helps to improve the plasticity and fatigue resistance of the alloy. The flux purification method, which focuses on impurity removal, can provide a reference for the actual impurity removal in aluminum alloy smelting.

Deepening the Understanding of Carbon Active Sites for ORR Using Electrochemical and Spectrochemical Techniques.

Defect-containing carbon nanotube materials were prepared by subjecting two commercial multiwalled carbon nanotubes (MWCNTs) of different purities to purification (HCl) and oxidative conditions (HNO3) and further heat treatment to remove surface oxygen groups. The as-prepared carbon materials were physicochemically characterized to observe changes in their properties after the different treatments. TEM microscopy shows morphological modifications in the MWCNTs after the treatments such as broken walls and carbon defects including topological defects. This leads to both higher surface areas and active sites. The carbon defects were analysed by Raman spectroscopy, but the active surface area (ASA) and the electrochemical active surface area (EASA) values showed that not all the defects are equally active for oxygen reduction reactions (ORRs). This suggests the importance of calculating either ASA or EASA in carbon materials with different structures to determine the activity of these defects. The as-prepared defect-containing multiwalled carbon nanotubes exhibit good catalytic performance due to the formation of carbon defects active for ORR such as edge sites and topological defects. Moreover, they exhibit good stability and methanol tolerances. The as-prepared MWCNTs sample with the highest purity is a promising defective carbon material for ORR because its activity is only related to high concentrations of active carbon defects including edge sites and topological defects.

A Scalable and Surfactant-Free Emulsion Method for Producing Microbeads from Varied Biomass Feedstocks.

Despite national and international regulations, plastic microbeads are still widely used in personal care and consumer products (PCCPs). These exfoliants and rheological modifiers cause significant microplastic pollution in natural aquatic environments. Microbeads from nonderivatized biomass like cellulose and lignin can offer a sustainable alternative to these nondegradable microplastics, but processing this biomass into microbeads is challenging due to limited viable solvents and high biomass solution viscosities. To produce biomass microbeads of the appropriate size range for PCCPs (ca. 200-800 μm diameter) with shapes and mechanical properties comparable to those of commercial plastic microbeads, we used a surfactant-free emulsion/precipitation method, mixing biomass solutions in 1-ethyl-3-methylimidazolium acetate (EMImAc) with various oils and precipitating with ethanol. While yield of microbeads within the target size range highly depends on purification conditions, optimized protocols led to >90% yield of cellulose microbeads. Kraft lignin was then successfully incorporated into beads at up to 20 wt %; however, higher lignin contents result in emulsion destabilization unless surfactant is added. Finally, the microbead shape and surface morphology can be tuned using oils of varying viscosities and interfacial tensions. Dripping measurements and pendant drop tensiometry confirmed that the higher affinity of cellulose for certain oil/IL interfaces largely controlled the observed surface morphology. This work thus outlines how biomass composition, oil viscosity, and interfacial properties can be altered to produce more sustainable microbeads for use in PCCPs, which have desirable mechanical properties and can be produced over a wide range of shapes and surface morphologies.

Unveiling the Catalytic Roles of DsBBS1 and DsBBS2 in the Bibenzyl Biosynthesis of Dendrobium sinense.

Dendrobium sinense, an endemic medicinal herb in Hainan Island, is rich in bibenzyl compounds. However, few studies have explored the molecular mechanisms of bibenzyl biosynthesis. This study presents a comprehensive analysis of DsBBS1 and DsBBS2 function in D. sinense. A molecular docking simulation revealed high-resolution three-dimensional structural models with minor domain orientation differences. Expression analyses of DsBBS1 and DsBBS2 across various tissues indicated a consistent pattern, with the highest expression being found in the roots, implying that they play a pivotal role in bibenzyl biosynthesis. Protein expression studies identified optimal conditions for DsBBS2-HisTag expression and purification, resulting in a soluble protein with a molecular weight of approximately 45 kDa. Enzyme activity assays confirmed DsBBS2's capacity to synthesize resveratrol, exhibiting higher Vmax and lower Km values than DsBBS1. Functional analyses in transgenic Arabidopsis demonstrated that both DsBBS1 and DsBBS2 could complement the Atchs mutant phenotype. The total flavonoid content in the DsBBS1 and DsBBS2 transgenic lines was restored to wild-type levels, while the total bibenzyl content increased. DsBBS1 and DsBBS2 are capable of catalyzing both bibenzyl and flavonoid biosynthesis in Arabidopsis. This study provides valuable insights into the molecular mechanisms underlying the biosynthesis of bibenzyl compounds in D. sinense.

Generation of densely labeled oligonucleotides for the detection of small genomic elements

The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.

Self-flocculating Chlorella vulgaris: A high-efficiency purification mechanism of radioactive Th4+ in an aquatic environment

This study aimed to investigate the purification of radioactive thorium (Th4+) by Chlorella vulgaris in aquatic environments. Single-factor experiments and response surface optimization tests identified optimal purification conditions. The purification and metabolic response mechanisms of Chlorella to Th4+ were elucidated using physiological and biochemical analyses, three-dimensional excitation-emission matrix (3D-EEM) analysis, and metabolomic profiling. Increases in the Th4+ concentration caused Chlorella to self-flocculate, significantly improving the Th4+ purification efficiency. Under optimal conditions, the Th4+ purification efficiency for Th4+ in wastewater by Chlorella stabilized between 94.3 % and 98.2 %. Morphological analysis revealed that the purified Th4+ existed mainly in a stable residual state. Chlorella efficiently purified wastewater during treatment by regulating environmental pH, performing redox reactions, and utilizing extracellular polymeric substances (EPS) to interact with Th4+. Metabolomic analysis indicated that Chlorella adapted to the Th4+-contaminated environment and enhanced its purification function by adjusting the synthesis of metabolites, such as carbohydrates, nucleotides, and amino acids. Chlorella demonstrated a remarkable self-flocculation phenomenon and a high-efficiency purification capability for Th4+, offering new possibilities for environmental remediation. Its purification mechanism involves environmental regulation, redox reactions, and complex metabolic adjustments. The results presented here provide theoretical support for environmental remediation using Chlorella.

Predictions of Chromatography Methods by Chemical Structure Similarity to Accelerate High-Throughput Medicinal Chemistry.

We introduce a new workflow that relies heavily on chemical quantitative structure-retention relationship (QSRR) models to accelerate method development for micro/mini-scale high-throughput purification (HTP). This provides faster access to new active pharmaceutical ingredients (APIs) through high-throughput experimentation (HTE). By comparing fingerprint structural similarity (e.g., Tanimoto index) with small training data sets containing a few hundred diverse small molecule antagonists of a lipid metabolizing enzyme, we can predict retention time (RT) of new compounds. Machine learning (ML) helps to identify optimal separation conditions for purification without performing the traditional crude QC step involving ultrahigh performance liquid chromatography (UHPLC) analyses of each compound. This green-chemistry approach with the use of predictive tools reduces cost and significantly shortens the design-make-test (DMT) cycle of new drugs by way of HTE.

Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro

BackgroundEnamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent.ResultsIn the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml.ConclusionsThese findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.