-
The Journal of Biological Chemistry Feb 2024Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers,...
Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.
Topics: Animals; Cattle; Adenosine Triphosphate; Dimerization; Mitochondria, Heart; Mitochondrial Membranes; Mitochondrial Proton-Translocating ATPases; Centrifugation, Density Gradient
PubMed: 38159856
DOI: 10.1016/j.jbc.2023.105603 -
Protein Expression and Purification Jul 2023The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification...
The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.
Topics: Animals; Antibodies, Monoclonal; Chromatography, Affinity; Chromatography, High Pressure Liquid; Staphylococcal Protein A; Drug Discovery
PubMed: 37023994
DOI: 10.1016/j.pep.2023.106269 -
Toxicon : Official Journal of the... May 2024The present work is carried out to protein isolation, purification, and characterization from leaves, stem, and seed of C. procera and to evaluate the larvicidal...
The present work is carried out to protein isolation, purification, and characterization from leaves, stem, and seed of C. procera and to evaluate the larvicidal potential on Anopheles stephensi. The whole protein was isolated using protein extraction buffer and precipitated by ammonium sulphate and larvicidal active protein was purified by the column chromatography. The homogeneity of larvicidal protein was confirmed by the SDS-PAGE. The identification of protein was done by the HPLC and LC-MS/ESI-MS. The crude protein from leaves showed 100% mortality of 3 instar larvae of An. stephensi at the concentration of 5.5 mg/ml after 24 h of exposure. The crude protein from stem showed 25% mortality and no mortality observed was observed in seed protein. The leaves crude protein was further purified by ion exchange chromatography and eluted fractions were tested for larvicidal potential. The purified single protein fractions L2 and L3 from C. procera leaves showed 100% mortality at concentration of 0.06 mg/ml. The homogeneity of purified protein was confirmed by SDS-PAGE and two bands of 26 kDa and 15 kDa protein were observed. The peptide sequence "R.SQMLENSFLIENVMKR.L" was identified in the trypsin digested homogenous protein fraction L2 and "R.DRGSQKR.N" peptide sequence in L3 fraction by LC-MS/ESI-MS. The CprL2 peptide showed the sequence similarity with the protein maturase K and CprL3 peptide showed the sequence similarity with ribosomal protein L20 of C. procera. The conserved functional domain was also identified in both the CprL2 and CprL3 peptide. The identified proteins showed strong larvicidal efficacy at very low concentration. The identified proteins are novel and natural larvicidal agents against An. stephensi and hence can be used to control the malaria.
Topics: Anopheles; Animals; Plant Leaves; Larva; Insecticides; Ribosomal Proteins; Plant Proteins; Calotropis; Amino Acid Sequence
PubMed: 38626820
DOI: 10.1016/j.toxicon.2024.107714 -
Journal of Agricultural and Food... Jun 2024This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as... (Review)
Review
This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as protein-rich fractions, protein concentrates, and isolates. ANFs mainly include lectins, trypsin inhibitors, phytic acid, phenolic compounds, oxalates, saponins, tannins, and cyanogenic glycosides. The current technologies used to recover proteins (e.g., wet extraction, dry fractionation) and novel technologies (e.g., membrane processing) are included in this review. The mechanisms involved during protein extraction/recovery that may enhance or decrease the ANF content in plant protein ingredients are discussed. However, studies on the effects of protein extraction/recovery on specific ANFs are still scarce, especially for novel technologies such as ultrasound- and microwave-assisted extraction and membrane processing. Although the negative effects of ANFs on protein digestibility and the overall absorption of plant proteins and other nutrients are a health concern, it is also important to highlight the potential positive effects of ANFs. This is particularly relevant given the rise of novel protein ingredients in the market and the potential presence or absence of these factors and their effects on consumers' health.
Topics: Animals; Chemical Fractionation; Nutritive Value; Plant Proteins; Trypsin Inhibitors; Humans
PubMed: 38780067
DOI: 10.1021/acs.jafc.4c00380 -
The Journal of Biological Chemistry Nov 2023Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein...
Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3C protease, and 3D RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3C. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABC becomes 20-fold more active than 3C in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.
Topics: Peptide Hydrolases; Poliovirus; Polyproteins; RNA, Viral; Viral Proteins; Protein Folding; Circular Dichroism; Protein Stability; Enzyme Activation; Protein Structure, Secondary; Amino Acid Sequence
PubMed: 37717698
DOI: 10.1016/j.jbc.2023.105258 -
Methods in Molecular Biology (Clifton,... 2024Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be...
Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be extracted from plant tissues, which are rather complex than other types of biological material. Different protocols have been applied depending on the protein source, such as seeds, pollen, leaves, roots, and flowers. Total protein amounts must also be determined before conducting gel electrophoresis. The most common methodologies include PAGE under native or denaturing conditions. Both procedures are used consequently for protein identification and characterization via mass spectrometry. Additionally, various staining procedures are available to visualize protein bands in the gel, facilitating the software-based digital evaluation of the gel through image acquisition.
Topics: Electrophoresis, Polyacrylamide Gel; Plant Proteins; Plants; Proteomics; Software; Staining and Labeling; Mass Spectrometry
PubMed: 38656496
DOI: 10.1007/978-1-0716-3778-4_18 -
Scientific Reports Jun 2024Broad-spectrum biocatalysts enzymes, Laccases, have been implicated in the complete degradation of harmful pollutants into less-toxic compounds. In this study, two...
Broad-spectrum biocatalysts enzymes, Laccases, have been implicated in the complete degradation of harmful pollutants into less-toxic compounds. In this study, two extracellularly produced Laccases were purified to homogeneity from two different Ascomycetes spp. Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12). The purified enzymes are monomeric units, with a molecular mass of 44 kDa and 68.7 kDa for TlFLU1 and TpFLU12, respectively, on SDS-PAGE and zymogram. It reveals distinct properties beyond classic protein absorption at 270-280 nm, with TlFLU1's peak at 270 nm aligning with this typical range of type II Cu site (white Laccase), while TpFLU12's unique 600 nm peak signifies a type I Cu site (blue Laccase), highlighting the diverse spectral fingerprints within the Laccase family. The K and k values revealed that ABTS is the most suitable substrate as compared to 2,6-dimethoxyphenol, caffeic acid and guaiacol for both Laccases. The bioinformatics analysis revealed critical His, Ile, and Arg residues for copper binding at active sites, deviating from the traditional two His and a Cys motif in some Laccases. The predicted biological functions of the Laccases include oxidation-reduction, lignin metabolism, cellular metal ion homeostasis, phenylpropanoid catabolism, aromatic compound metabolism, cellulose metabolism, and biological adhesion. Additionally, investigation of degradation of polycyclic aromatic hydrocarbons (PAHs) by purified Laccases show significant reductions in residual concentrations of fluoranthene and anthracene after a 96-h incubation period. TlFLU1 Laccase achieved 39.0% and 44.9% transformation of fluoranthene and anthracene, respectively, while TpFLU12 Laccase achieved 47.2% and 50.0% transformation, respectively. The enzyme structure-function relationship study provided insights into the catalytic mechanism of these Laccases for possible biotechnological and industrial applications.
Topics: Talaromyces; Laccase; Trichoderma; Fungal Proteins; Substrate Specificity; Copper; Kinetics; Oxidoreductases; Catalytic Domain
PubMed: 38862560
DOI: 10.1038/s41598-024-63959-z -
Journal of Chromatography. A May 20243D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical...
3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time ∼100 mL/h, resolution ∼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed >85 % IgG recovery from harvested cell culture fluid with purities above 98 %. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO functionalised columns employed for polishing achieved removal of leaked Protein A (down to 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.
Topics: Printing, Three-Dimensional; Antibodies, Monoclonal; Staphylococcal Protein A; Immunoglobulin G; Cricetulus; CHO Cells; Chromatography, Affinity; Animals; Chromatography, Ion Exchange; Ink
PubMed: 38626540
DOI: 10.1016/j.chroma.2024.464873 -
Applied Biochemistry and Biotechnology Feb 2024Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to...
Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.
Topics: Animals; Rats; Granulocyte Colony-Stimulating Factor; Neutrophils; Polymers; Recombinant Proteins
PubMed: 37209276
DOI: 10.1007/s12010-023-04522-w -
Protein Expression and Purification Jul 2024Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV,...
Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.
Topics: Chlamydophila pneumoniae; Crystallography, X-Ray; Crystallization; Bacterial Proteins; Escherichia coli; Molecular Docking Simulation; Recombinant Proteins; Deoxyribonuclease IV (Phage T4-Induced); Cloning, Molecular
PubMed: 38521114
DOI: 10.1016/j.pep.2024.106476