Metal Affinity Chromatography Research Articles

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.

Scar-free tag removal by CASPON® enzyme with broad physicochemical stability in biomanufacturing – A case study of five proteins

Manufacturing of recombinant proteins in microbial systems, in particular in E. coli, generally requires thorough process development due to the absence of a platform process. The caspase-based fusion process (CASPON®) offers a platform manufacturing process using special protease cleavable fusion-tags. These tags allow the implementation of His-tag based affinity chromatography for facile target protein capture and offer expression and solubility enhancing capabilities. The tags are intended as N-terminal fusion motifs that can be fully cleaved using a modified caspase-2 protease, the CASPON® enzyme. Here, we systematically explore the influence of various physicochemical parameters on its enzymatic activity. This characterization was performed in parallel in two independent research laboratories using different assays, i.e. a Förster resonance energy transfer-based assay using small peptide substrates and a reversed phase high performance liquid chromatography method using model proteins. Both assays demonstrated great agreement and reveal that CASPON® enzyme is highly active at a wide range of temperatures, pH and is resistant to a variety of chemical substances that are commonly employed in bioprocessing, e.g. NaCl, kosmotropes, chaotropes and others. The presence of imidazole at concentrations commonly found in the elution fraction of immobilized metal affinity chromatography does not seem to affect the activity of CASPON® enzyme, enabling its use directly after the capture step in downstream processing (DSP). A case study of five biopharmaceuticals is presented. The platform process exhibits consistently high performance in up- and downstream processing, achieving high soluble titers, high yield and purity. The presence of host cell proteins during the DSP in particular was investigated in-depth using process proteomics, revealing that a core of host cell proteins is process dependent and can commonly be expected to be found in the platform DSP. The information presented here can serve as a guide on how to implement the CASPON® platform process for the production of various recombinant proteins.

Aggregation-Dispersion Chromatography: Application of Elastin-like Polypeptides

Protein purification is a crucial step for various downstream applications like drug development, antibody preparation, and structure determination. The constant pursuit is for methods that are more efficient and cost-effective. We propose a novel approach using an elastin-like polypeptide (ELP) as an aggregation core that serves as an anchor between the beads in a chromatography column. In this method, a chilled sample containing a [target protein type] fusion protein is loaded onto a pre-equilibrated IMAC (immobilized metal affinity chromatography) column with a low-salt buffer. The column is then washed with a warm buffer containing high salt to remove impurities. Here, the key step involves warming the column above the ELP’s transition temperature (Tt), which triggers its aggregation. This aggregation is expected to trap the target protein tightly between the beads. Subsequently, a harsh wash with high salt and high imidazole can be applied to remove even persistent contaminants, achieving high protein purity. Finally, the temperature is lowered, and a cold, low-salt buffer is introduced to reverse the aggregation and elute the purified target protein. This method has the potential to eliminate the need for sophisticated chromatography systems while still achieving high protein purity.

Polymerization potential of a bacterial CotA-laccase for β-naphthol: enzyme structure and comprehensive polymer characterization.

Laccases are blue-multicopper containing enzymes that are known to play a role in the bioconversion of recalcitrant compounds. Their role in free radical polymerization of aromatic compounds for their valorization remains underexplored. In this study, we used a pBAD plasmid containing a previously characterized CotA laccase gene (abbreviated as Bli-Lacc) from Bacillus licheniformis strain ATCC 9945a to express this enzyme and explore its biotransformation/polymerization potential on β-naphthol. The protein was expressed from TOP10 cells of Escherichia coli after successful transformation of the plasmid. Immobilized metal affinity chromatography (IMAC) was used to generate pure protein. The biocatalytic polymerization reaction was optimized based on temperature, pH and starting enzyme concentration. 1H and 13C solution nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and solid-state NMR (ssNMR) were used to characterize the formed polymer. A one-gram conversion reaction was done to explore applicability of the reaction in a pilot-scale. The polymerization reaction generated a brown precipitate, and its chemical structure was confirmed using 1H and 13C NMR and FTIR. SsNMR revealed the presence of two different orientational hydroxyl functional groups in the polymer in addition to the presence of a very small amount of ether linkages (< 2%). This analysis elucidated that polymerization occurred mainly on the carbons of the aromatic rings, rather than on the carbons attached to the hydroxyl groups, resulting in a condensed ring or polynuclear aromatic structure. The reaction was optimized, and the highest yield was attained under conditions of 37°C, pH 10 and a starting enzyme concentration of 440 nM in 50 mM phosphate buffer. A one-gram conversion yielded 216 mg of polymer as dry mass. The crystal structure of the enzyme was solved at 2.7 Å resolution using X-ray crystallography and presented with a hexagonal space group. The final structure was deposited in the Protein Databank (PDB) with an ID-9BD5. This article provides a green/enzymatic pathway for the remediation of phenolics and their valorization into potential useful polymeric materials. The comprehensive analysis of the formed polymer provides insight into its structure and functional moieties present. Based on the yield of the one-gram conversion, this synthetic method proves useful for a pilot-scale production level and opens opportunities to invest in using this polymer for industrial/environmental applications.

A INNOVATE APPROACH TO METAL REMOVAL IN CRYOGELS USING ELECTRICAL POTENTIAL IN A FIXED BED SYSTEM

The use of chelating agents in affinity chromatography poses significant challenges due to their tendency to form chemical bonds with metals. During the purification process, these agents can dissociate from the matrix, potentially contaminating the purified biomolecule and rendering it unsuitable for therapeutic or other biological applications. This study introduces an innovative approach for the electroadsorption of Cu²⁺ ions onto polyacrylamide (PAam) cryogels using a fixed-bed system under the application of electrical potential. This method reduces the risk of metal contamination by eliminating the need for chelating agents commonly used in immobilized metal ion affinity chromatography (IMAC). The cryogels demonstrated excellent structural integrity, retaining their shape after compression and rehydration. Electroadsorption experiments revealed that a minimum voltage of 3V was required to establish effective removal sites for Cu²⁺ ions, with a maximum removal capacity of approximately 19 mg/g (PAam)". At higher voltages (3V and 5V), the electroadsorbed ions were efficiently desorbed using ethylenediaminetetraacetic acid (EDTA) solution. The distinct d⁹ electron configuration of Cu²⁺ makes it particularly prone to forming coordinated bonds with the lone pairs of N present in the PAam structure. The study also highlights the impact of flow rates on saturation times, with faster rates leading to quicker saturation without compromising removal efficiency. These results demonstrate that electroadsorption in PAam cryogels is a promising alternative to IMAC for metal ion purification in chromatographic applications, providing a cleaner and more cost-effective solution.

Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is one of the most important post-translational modifications that regulates many biological processes. There have been relatively few studies on the phosphoproteome of recombinant Chinese hamster ovary (CHO) cells to date despite phosphorylation playing a crucial role in regulating many molecular and cellular processes relevant to bioprocess phenotypes including, for example, transcription, translation, growth, apoptosis, and signal transduction. In this chapter, we provide a protocol for phosphoproteomic analysis of CHO cells using phosphopeptide enrichment with metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) techniques, followed by site-specific identification of phosphorylated residues using liquid chromatography mass spectrometry (LC-MS), multistage activation (MSA), and MS3 strategies. This protocol can also be used for quantitative phosphoproteomic analysis using both labeled and label-free approaches.

Production of anti-rM14 and anti-rM42 antibodies and their application in the stidy of expression of metalloproteases M14 and M42 of keratinolytic strain of Bacillus paralicheniformis

In the present study, two metalloproteases M14 and M42 from a Kazakh strain of Bacillus paralicheniformis T7 with high proteolytic activity were successfully cloned, expressed and purified. Protease genes were amplified by polymerase chain reaction (PCR) using specific primers selected on the basis of full genome sequencing data of B. paralicheniformis strain N7 (GenBank accession #CP124861). The amplified fragments were cloned in the expression vector pET-28c(+). Recombinant analogs of the metalloproteases M14 and M42 were obtained by plasmid expression in Escherichia coli cells of the strain ArcticExpressRP(DE3). Using metal affinity chromatography, rM14 and rM42 proteins were purified and polyclonal antibodies were produced by immunization of rabbits. Using anti-rM14 and anti-rM42 antibodies, the expression level of native metalloproteases M14 and M42 in cell lysate and culture fluid of B. paralicheniformis T7 strain was examined after culturing the strain on keratin-containing media: chicken feather, horn, hoof, hide and wool of cattle. Substrate-dependent expression of metalloprotease genes of B. paralicheniformis strain T7 was established. The obtained results indicate the prospective use B. paralicheniformis strain T7 as a producer of metalloproteases M14 and M42.

Overexpression, purification and biochemical studies of Sortase A from Enterococcus faecalis (Ef) and its inhibition studies with Aloenin

Sortase A (SrtA) is a bacterial transpeptidase that garnishes the bacterial surface by adding various virulent factors or proteins by cleaving the LPXTG-specific motif between T and G amino acids. These virulence factors assist in the attachment of host cells, which are essential for bacterial virulence. Enterococcus species are among the multidrug-resistant bacteria that cause nosocomial infections; they have drawn a lot of attention recently. SrtA from E. faecalis (Ef) plays a critical role in pathogenesis, making it a suitable target for the development of antibacterial agents. Since SrtA is not involved in bacterial growth and is present on the surface of bacteria, the probability of developing antibiotic resistance is minimal. In this work, we have done cloning, expression and purification of Ef-SrtA using IMAC (Immobilised Metal Affinity Chromatography) followed by Gel filtration chromatography. Purified Ef-SrtA showed maximum activity at pH-8 and temperature between 45 and 55 °C. The fluorescent assay for kinetic studies of Ef-SrtA showed Vmax 3.852 µM.min−1 and kcat 7.7 × 10−2s−1 for the hydrolysis of substrate using Abz-LPETG-K(Dnp)-NH2. We have selected fifteen Aloe vera extracted compounds and performed virtual screening and docking experiments to identify potential inhibitors against Ef-SrtA. Among fifteen molecules, Aloenin-a which was bound to the active site with a binding energy of -6.1 kcal/mol, interacted with the active site residues, Arg139, Pro105, Leu39, Ala46, and Cys126. Aloenin-a showed a significant inhibitory effect against Ef-SrtA, with an IC50 value of 20.68 µM. Aloenin-a inhibits biofilm formation at concentrations of 20–250 µg/mL. The fibrinogen assay showed adherence to fibrinogen was reduced in the presence of Aloenin-a for E. faecalis. The results demonstrated that Aloe vera extracts containing Aloenin-a can be a significant antagonist of Ef-SrtA.

Isolation and characterisation of zinc‐chelating peptides from tilapia (Oreochromis niloticus) protein hydrolysate

SummaryThis study aimed to isolate and characterise zinc‐chelating peptides from tilapia (Oreochromis niloticus) protein hydrolysate. The hydrolysate was fractionated using a multi‐ultrafiltration technique, yielding peptide fractions with molecular weights between 0.5 and 5 kDa. These fractions were further purified using immobilised metal ion affinity chromatography (IMAC‐Zn2+) and reverse high‐performance liquid chromatography (RP‐HPLC). Among the fractions obtained, the peptide fraction F211 exhibited a zinc‐binding capacity of 76.26 ± 2.46 mg g−1. Sequence analysis indicated that zinc binding occurred at amino acids on the F21 peptide and at valine or glutamate residues on the F22 peptide fraction. Structural characterisation revealed that amino nitrogen and oxygen atoms from carboxylate groups were the primary binding sites for Zn2+. Additionally, the zinc–peptide complex demonstrated strong thermal stability, which is significant for potential applications in food processing and storage, where maintaining the integrity of nutritional supplements under varying temperature conditions is crucial. These findings provide a feasible approach for the separation and purification of zinc‐binding peptides from tilapia protein hydrolysates and contribute to the understanding of the binding mechanisms between zinc and peptides.

189 Association of the liver phosphoproteome with divergent dry matter intake in finishing beef steers

Abstract As the need for improving the sustainability of the beef industry increases, optimizing feed efficiency is of the utmost importance. The liver has a central role in nutrient metabolism, and for ruminants particularly, it is essential for providing glucose through gluconeogenesis via substrates such as propionate, lactate, and glycerol. Further, through cell signaling and vagal afferent signals, the liver acts to stimulate and inhibit dry matter intake (DMI). Thus, the objective of this study was to investigate alterations in the phosphoproteome of finishing beef steers in relation to divergent DMI to identify changes in the abundance of signaling proteins. Angus steers [n = 57; initial body weight (BW) = 518 ± 27 kg] underwent a 63-d intake trial utilizing an Insentec Roughage Intake Control System (Hokofarm Group, Emmeloord, The Netherlands). Prior to beginning the trial, liver biopsy samples were collected from all steers. Samples from steers with the greatest and least DMI (n = 8 each) were utilized for phosphoproteomic analysis. Liver protein was isolated and labeled with isobaric tandem mass tagging reagents. Samples were enriched for phosphopeptides using immobilized metal affinity chromatography and subsequently analyzed using high-performance liquid chromatography tandem mass spectrometry. Spectral data were searched in the National Center for Biotechnology Information Bos taurus protein database using MaxQuant (version 2.2.0.0; Max Planck Inst., Munich, Germany), and the data were analyzed using Perseus (version 2.0.10.0; Max Planck Inst.). Two sample t-tests were conducted to determine the difference in phosphopeptide abundance between steers with high and low DMI. A total of 16,365 phosphopeptides were identified, and 287 were differentially expressed (False Discovery Rate &amp;lt; 0.05). In addition, 2,777 phosphopeptides were differentially expressed based on an unadjusted P &amp;lt; 0.05 which were used for enrichment analysis of Gene Ontology processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the Database for Annotation, Visualization, and Integrated Discovery. The background for this analysis was all identified phosphopeptides. Gene ontology terms enriched included processes related to phosphorylation activity, intracellular signal transduction, response to insulin, kinase activity, and signaling protein binding. Enriched KEGG pathways included pathways related to insulin signaling, such as AMPK signaling, mTOR signaling, and MAPK signaling. Further, pathways related to glycolysis and gluconeogenesis, glucagon signaling, insulin resistance, carbon metabolism, and various other signaling pathways were also enriched. These results suggest a link between the liver phosphoproteome and DMI, indicating that divergent DMI is associated with alterations in signaling pathways related to nutrient uptake and metabolic processes.

Cytotoxic T-lymphocyte antigen 4 and programmed cell death ligand 1 blockade increases the effectiveness of interleukin-15 immunotherapy in a bovine leukemia model.

Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4- and PD-L1-blocking algorithm. The codon-optimized bIL15 gene was synthesized under de novo conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent Escherichia coli BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the Bcl2, STAT3, and STAT5 genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells. Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the Bcl2, STAT3, and STAT5 genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production. The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections.

Cloning, Expression, and Characterization of a Metalloprotease from Thermophilic Bacterium Streptomyces thermovulgaris.

Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6-8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries.

Comparative quantitative phosphoproteomic and parallel reaction monitoring analysis of soybean roots under aluminum stress identify candidate phosphoproteins involved in aluminum resistance capacity

Aluminum (Al) toxicity adversely impacts soybean (Glycine max) growth in acidic soil. Reversible protein phosphorylation plays an important role in adapting to adverse environmental conditions by regulating multiple physiological processes including signal transduction, energy coupling and metabolism adjustment in higher plant. This study aimed to reveal the Al-responsive phosphoproteins to understand their putative function and involvement in the regulation of Al resistance in soybean root. We used immobilized metal affinity chromatography to enrich the key phosphoproteins from soybean root apices at 0, 4, or 24 h Al exposure. These phosphoproteins were detected using liquid chromatography-tandem mass spectrometry measurement, verified by parallel reaction monitoring (PRM), and functionally characterized via overexpression in soybean hairy roots. A total of 638 and 686 phosphoproteins were identified as differentially enriched between the 4-h and 0-h, and the 24-h and 0-h Al treatment comparison groups, respectively. Typically, the phosphoproteins involved in biological processes including cell wall modification, and RNA and protein metabolic regulation displayed patterns of decreasing enrichment (clusters 3, 5 and 6), however, the phosphoproteins involved in the transport and metabolic processes of various substrates, and signal transduction pathways showed increased enrichment after 24 h of Al treatment. The enrichment of phosphoproteins in organelle organization bottomed after 4 h of Al treatment (cluster 1). Next, we selected 26 phosphoproteins from the phosphoproteomic profiles, assessed their enrichment status using PRM, and detected enrichment patterns similar to those observed via phosphoproteomic analysis. Among them, 15 phosphoproteins were found to reduce the accumulation of Al and callose in Al-stressed soybean root apices when their corresponding genes were individually overexpressed in soybean hairy roots. In summary, the findings of this study facilitated a comprehensive understanding of the protein phosphorylation events involved in Al resistance responses and revealed some critical phosphoproteins that enhance Al resistance in soybean roots.

Biotechnological Production of the Recombinant Two-Component Lantibiotic Lichenicidin in a Bacterial Expression System

Objective: Lantibiotics are a family of bacterial antimicrobial peptides synthesized by ribosomes, that undergo post-translational modification to form lanthionine (Lan) and methyllanthionine (MeLan) residues. Lantibiotics are currently considered promising agents for combating antibiotic-resistant bacterial infections. This paper presents a biotechnological method for obtaining two components of the lantibiotic lichenicidin from Bacillus licheniformis B-511, Lchα and Lchβ. Such a system has the potential to facilitate the production of not only lichenicidin, but also other lantibiotics, including two-component ones, and also to enable the study of their biosynthesis and the activity and substrate specificity of modifying enzymes. Methods: The developed system is based on heterologous coexpression of the genes of Lchα and Lchβ precursors with the genes of their corresponding modifying enzymes in the cytoplasm of Escherichia coli BL21(DE3). Subsequent steps included immobilized metal affinity chromatography of the His-tagged hybrid peptide under denaturing conditions, cyanogen bromide cleavage in acidic medium, and final purification using reverse-phase HPLC. Results and Discussion: The system was employed for the expression and purification of lantibiotics, resulting in the successful isolation of the β-component of lichenicidin in high yield (approximately 4 mg/L of culture). This purified beta component exhibited structural and functional characteristics comparable to its natural counterpart, which was purified from the natural producer. However, the yield of the mature α-component of lichenicidin in such a system was significantly lower. Conclusions: The work presents a biotechnological method for obtaining recombinant two-component lantibiotic lichenicidin, which has proven to be particularly effective in the case of Lchβ. The developed method can also be applied to the production of other promising lantibiotics and their further research.

Ice recrystallization inhibition activity of pulse protein hydrolysates after immobilized metal affinity separation

Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins.

Exploitation of porphyrin-based titanium-rich porous organic polymers for targeted phosphopeptide enrichment from the serum of colorectal cancer individuals.

Aporphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol),highselectivity (β-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.

Methanolic extracts of litchi (Litchi chinensis Sonn.): A novel approach of targeting glucose-6-phosphate dehydrogenase for liver cancer therapy

Cancer metabolism has emerged as a potential target for innovative therapeutic approaches in the treatment of cancer. Cancer metabolism has received much attention, particularly in relation to glucose metabolism. It has been observed that human malignancies have high levels of glucose-6-phosphate dehydrogenase (G6PD) activity which is an important enzyme of glucose metabolism. This overactivity is associated with the cell death and angiogenesis, highlighting its potential as a viable target for cancer treatment. This study was conducted to examine the methanolic extracts from the seeds, bark and leaves of litchi (Litchi chinensis Sonn.) in order to discover effective compounds targeting G6PD and potentially active entities against liver cancer. Plant extract screening for the target protein was carried out through enzymatic activity assay. The recombinant plasmid pET-24a-HmG6PD was expressed in E. coli (BL21-DE3) strain, then purified and assessed using metal affinity chromatography with Ni-NTA columns and SDS-PAGE. The cytotoxicity of plant extracts against liver cancer HepG2 cells was assessed using the MTT assay. All three extracts demonstrated significant inhibitory effects (>80% inhibition) against G6PD. They were then subjected to testing at various concentrations, and their IC50 values were subsequently determined. The extracts of litchi (leaf, IC50: 1.199 μg/mL; bark, IC50: 2.350 μg/mL; seeds, IC50: 1.238 μg/mL) displayed significant inhibition of G6PD activity at lower concentrations. Subsequently, the leaf extract of litchi was further assessed for its impact on HepG2 cell lines in a dose-dependent manner and exhibited strong potential as an inhibitor of cancer cell progression. Moreover, the results of acute toxicity study in mice revealed nontoxic effects of litchi leaf extract on hepatocytes. The results imply that Litchi chinensis leaf extract could be considered as a promising candidate for safer drug development in the treatment of liver cancer.

Production, purification, and quality assessment of borrelial proteins CspZ from Borrelia burgdorferi and FhbA from Borrelia hermsii

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH15-20 and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF.Key points• DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment• Borrelia spp. possesses immune evasion mechanisms, including human host complement• CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1Graphical

Evaluating Imide-Based Mass Spectrometry-Cleavable Cross-Linkers for Structural Proteomics Studies.

Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas phase. In this study, we further advance the structural capabilities of DSBU by remodeling the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl disuccinic imide (DSSI) as a prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography enrichment. We detail DSSI synthesis and describe its behavior in solution and in the gas phase while cross-linking isolated proteins and human cell lysates. DSSI and DSBU cross-links are compared at the same enrichment depth to bridge these two cross-linker classes. We validate DSSI cross-links by mapping them in high-resolution structures of large protein assemblies. The cross-links observed yield insights into the morphology of intrinsically disordered proteins and their complexes. The DSSI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers.

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.