As the need for antibody production rises, there is an urgent need to lower the costs and enhance the efficiency of the separation process. Currently, the chromatographic media used for antibody separation and purification often focus on individual properties of antibodies, such as affinity, hydrophobicity, and charge, leading to issues like low purification efficiency or inadequate adsorption capacity. To address this, an electrostatically coupled polypeptide affinity medium (FD7-3, 5-diaminobenzoic acid n-sepharose, FD7-DA-Sepharose) was developed for rapid purification of antibodies from cell culture supernatant. This medium utilized 3, 5-diaminobenzoic acid as a spacer to attach the heptapeptide-affinity ligand (FYEILHD, FD7) to agarose microspheres. Antibodies could be adsorbed through charge interactions with the carboxyl functional group of the FD7-DA-Sepharose spacer, while FD7 enhanced electrostatic coupling and affinity adsorption through synergistic effects, significantly increasing the adsorption capacity while maintaining the affinity and specificity. The influences of pH and ionic strength on adsorption capacity were investigated with human immunoglobulin as a model protein. The static adsorption capacity (Q_m) of FD7-DA-Sepharose in the solution of pH 6.0 reached 67.73 mg/mL, representing a 52.68% increase compared with that (44.36 mg/mL) of the commercial Protein A affinity medium. Furthermore, the elution conditions for FD7-DA- Sepharose were mild (20 mmol/L PB, 0.5 mol/L NaCl, pH 6.0), in contrast to the harsh acidic elution (pH 2.7-3.6) typically associated with Protein A, which can damage antibody integrity. The FD7-DA-Sepharose medium was then employed to purify antibodies from cell culture supernatant, achieving the yield of 94.8% and the purity of 98.4%. The secondary structure of the purified antibody was determined by circular dichroism spectroscopy. The results demonstrated that FD7-DA-Sepharose enabled efficient purification of antibodies from cell culture supernatant, which provided a cost-effective solution (approximately one-third the price of commercial Protein A affinity medium) with gentle elution conditions that preserve the natural conformation of antibodies. This approach paves a novel, economical, and efficient way for the separation and purification of antibodies from cell culture supernatant.
Chromatography, Affinity/methods* ; Static Electricity ; Humans ; Sepharose/analogs & derivatives* ; Peptides/chemistry* ; Adsorption ; Antibodies/isolation & purification*

Two new isomeric modified tripeptides, aspergillamides C and D (compounds 1 and 2), together with fifteen known compounds (compounds 3-17), were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008. The structures of the new compounds, including absolute configurations, were determined by extensive analyses of spectroscopic data (NMR, MS, UV, and IR) and comparisons between the calculated and experimental electronic circular dichroism (ECD) spectra. Butyrolactone I (compound 11) exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with the IC being 5.11 ± 0.53 μmol·L, and acted as a noncompetitive inhibitor based on kinetic analysis.
4-Butyrolactone ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Animals ; Aspergillus ; chemistry ; Chemistry Techniques, Analytical ; Dipeptides ; chemistry ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Indoles ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Mycobacterium tuberculosis ; drug effects ; Peptides ; chemistry ; isolation & purification ; pharmacology ; Polyketides ; chemistry ; isolation & purification ; pharmacology ; Porifera ; microbiology ; Protein Tyrosine Phosphatases ; chemistry

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism

Two cyclopeptides, celogentin L (1) and its epimer lyciumin A (2) were firstly isolated from Celosia argentea L.. The planar structures of the two compounds were fully determined by spectroscopic data, including 1D-, 2D-NMR, and HR-ESI/MS. The absolute configurations of amino acid components were assigned via chiral-phase HPLC analyses after acid hydrolysis. Furthermore, the configuration of C-N linkage at the glycine Cα was elucidated by extensive analyses of 2D-NMR and comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Cytotoxicity of the two compounds against human alveolar epithelial A549, hepatocellular carcinoma HepG2, and cervical cancer Hela cell lines was assayed. Although both of them were inactive in these cells, the present findings add new facets for the chemistry of Celosia argentea.
A549 Cells ; Cell Survival ; drug effects ; Celosia ; chemistry ; Chemistry Techniques, Analytical ; HeLa Cells ; Hep G2 Cells ; Humans ; Molecular Conformation ; Molecular Structure ; Peptides, Cyclic ; chemistry ; isolation & purification ; toxicity ; Seeds ; chemistry ; Stereoisomerism

Herbal extracts have been extensively used worldwide for their application on memory improvement, especially among aged and memory-deficit populations. In the present study, the memory loss induced by human Abeta protein over-expression in fruitfly Alzheimer's disease (AD) model was rescued by multiple extracts from Gardenia jasminoides. Three extracts that rich with gardenia yellow, geniposide, and gardenoside components showed distinct rescue effect on memory loss. Further investigation on adding gardenoside into a formula of Ganoderma lucidum, Panax notoginseng and Panax ginseng (GPP) also support its therapeutic effects on memory improvement. Interestingly, the application of GPP and gardenoside did not alter the accumulation of Abeta proteins but suppressed the expression of immune-related genes in the brain. These results revealed the importance and relevancy of anti-inflammation process and the underlying mechanisms on rescuing memory deficits, suggesting the potential therapeutic use of the improved GPP formulation in improving cognition in defined population in the future.
Alzheimer Disease ; drug therapy ; Animals ; Antimicrobial Cationic Peptides ; genetics ; Brain ; drug effects ; immunology ; Cognition ; drug effects ; Disease Models, Animal ; Drosophila ; Drosophila Proteins ; genetics ; Gardenia ; chemistry ; Gene Expression Regulation ; drug effects ; Immunity, Innate ; drug effects ; Iridoids ; chemistry ; isolation & purification ; pharmacology ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Polymerase Chain Reaction

Amphibian skin contains rich bioactive peptides. Especially, a large amount of antimicrobial peptides have been identified from amphibian skin secretions. Antimicrobial peptides display potent cytolytic activities against a range of pathogenic bacteria and fungi and play important defense roles. No antimicrobial peptides have been reported from toads belonging to the family of Pelobatidae. In this work, two novel antimicrobial peptides (Megin 1 and Megin 2) were purified and characterized from the skin venoms of spadefoot toad Megophrys minor (Pelobatidae, Anura, Amphibia). Megin 1 had an amino acid sequence of FLKGCWTKWYSLKPKCPF-NH2, which was composed of 18 amino acid residues and contained an intra-molecular disulfide bridge and an amidated C-terminus. Megin 2 had an amino acid sequence of FFVLKFLLKWAGKVGLEHLACKFKNWC, which was composed of 27 amino acid residues and contained an intra-molecular disulfide bridge. Both Megin 1 and Megin 2 showed potential antimicrobial abilities against bacteria and fungi. The MICs of Megin 1 against Escherichia coli, Bacillus dysenteriae, Staphylococcus aureus, Bacillus subtilis, and Candida albicans were 25, 3, 6.25, 3, and 50 μg·mL(-1), respectively. The corresponding MICs for Megin 2 were 6.25, 1.5, 12.5, 1.5, and 12.5 μg·mL(-1), respectively. They also exerted strong hemolytic activity against human and rabbit red cells. The results suggested that megin peptides in the toad skin of M. minor displayed toxic effects on both eukaryotes and prokaryotes. This was the first report of antimicrobial peptides from amphibians belonging to the family of Pelobatidae.
Amino Acid Sequence ; Amphibian Venoms ; chemistry ; immunology ; isolation & purification ; Animals ; Anura ; immunology ; Bacillus ; Candida albicans ; Erythrocytes ; physiology ; Escherichia coli ; Female ; Hemolysis ; Humans ; Male ; Peptides ; chemistry ; immunology ; isolation & purification ; Rabbits ; Sequence Alignment ; Skin ; chemistry ; immunology ; Staphylococcus aureus

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.
Acetylglucosamine/*metabolism ; Amino Acid Sequence ; Animals ; Brain/*metabolism ; Chromatography, Affinity ; Glycosylation ; Mice ; Molecular Sequence Data ; Peptides/isolation & purification ; Phosphorylation ; Synapsins/chemistry/*metabolism ; Synaptosomes/*metabolism ; Tandem Mass Spectrometry ; tau Proteins/chemistry/*metabolism

A fraction named GFC-1 with high antioxidant activities in vitro was isolated from the enzymatic hydrolysates of roasted pills of Asini Corii Colla, and the peptides in this fraction were identified. The enzymatic hydrolysates were isolated and purified with anion exchange chromatography and Sephadex G-25 filtration chromatography successively. GFC-1, a fraction isolated from the hydrolysates, exhibited the highest DPPH and ABTS scavenging capacity (DPPH 47. 95% at 2.0 g x L(-1) and ABTS 97.20% at 0.40 g x L(-1). Nine peptides from GFC-1 were identified by LC-ESI-MS/MS coupled with TurboSEQUEST search software and Swiss-Prot data base, and a high repetition core sequence GPAGPP*GPP* was also found.
Animals ; Antioxidants ; chemistry ; isolation & purification ; Equidae ; Hydrolysis ; Mass Spectrometry ; Peptides ; chemistry ; isolation & purification ; Protein Hydrolysates ; chemistry ; Skin ; chemistry

To study the secondary metabolites of a marine-derived fungus Ascotricha sp. ZJ-M-5, several chromatographic methods including macroporous resin, silica gel, ODS and Sephadex LH-20 were used to isolate the compounds, and their structures were elucidated on the basis of physicochemical properties and spectroscopic methods. Ten compounds were obtained and identified as ascotrichic acid B (1), (3R)-6-hydroxymellein (2), beta-carboline (3), (22E, 24R)-ergosta-7, 22-diene-3beta, 5alpha, 6beta-triol (4), (22E, 24R)-ergosta-7, 22-diene-3beta, 5alpha, 6beta, 9alpha-tetraol (5), cyclo (Leu-Pro) (6), cyclo (Ile-Leu) (7), cyclo (Pro-Val) (8), cyclo (Pro-Gly) (9), and cyclo (Hpro-Ala) (10). Among them, compound 1 is a new 3, 4-seco-lanostane triterpenoid which has been isolated from the filamentous fungi for the first time, and compounds 2-10 are firstly isolated from Ascotricha genus.
Antineoplastic Agents ; chemistry ; isolation & purification ; pharmacology ; Ascomycota ; chemistry ; Carbolines ; chemistry ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dipeptides ; chemistry ; isolation & purification ; pharmacology ; Drug Screening Assays, Antitumor ; Humans ; Lanosterol ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Peptides, Cyclic ; chemistry ; isolation & purification ; pharmacology

OBJECTIVETo investigate the mechanisms for inhibition effects of PESV on proliferation of non-small cell lung cancer cell line A549.

METHODMTT was used to observe cell growth and proliferation of A549 at different concentrations of PESV. Flow cytometry (FCM) was applied to analyze cell cycle distribution. Immunocytochemistry and western blot assay was recruited to detect the expression of VEGF, HIF-1alpha, PTEN after the intervention of PESV.

RESULTA549 cells may be arrested mainly in G0/G1 phase and cell proliferation was significantly inhibited (P < 0.01) after PESV intervention in a certain range of concentration. PESV can significantly reduce the expression of HIF-1alpha,VEGF and increase the expression of PTEN.

CONCLUSIONPESV can block cell cycle and inhibit angiogenesis directly to inhibit cell proliferation of non-small cell lung cancer cell line A549 mainly through reducing the expression of HIF-1alpha, VEGF and increasing the expression of PTEN.

Antineoplastic Agents ; isolation & purification ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; PTEN Phosphohydrolase ; metabolism ; Peptides ; isolation & purification ; pharmacology ; Scorpion Venoms ; chemistry ; Vascular Endothelial Growth Factor A ; metabolism