Tricine-sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Purification and antimicrobial mechanism of a novel bacteriocin produced by Lactiplantibacillus plantarum FB-2

Lactiplantibacillus plantarum, a major producer of bacteriocins, is generally considered a safe microorganism. In this study, Plantaricin FB-2 was extracted from L. plantarum FB-2 and studied for antimicrobial mechanism against Staphylococcus aureus. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of Plantaricin FB-2 is 1–3.3 kDa. Plantaricin FB-2 is non-hemolytic and its minimum inhibitory concentration against S. aureus ATCC6538 is about 256 μg/mL. Plantaricin FB-2 exerts the antibacterial effect by destroying the structure of the bacterial cell membrane, increasing cell permeability, promoting the leakage of cytoplasmic contents (proteins, DNA, K+, Ca2+, etc.), and increasing intracellular reactive oxygen species levels. This study provides the theoretical basis for the development of L. plantarum produced Plantaricin FB-2 as a new antibacterial agent.

Identification and characterisation of a novel heptapeptide mackerel by-product hydrolysate, and its potential as a functional fertiliser component

Functional fertilisers for hydroponics are in great demand. Herein, we isolated peptides from mackerel by-products, a valuable source of bioactive peptides. The pellet-phase fraction obtained after cold-acetone extraction exhibited plant growth-promoting activity in wheat hydroponics, and the presumed peptides were determined to be ≤ 1 kDa based on molecular weight cut-off and tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Size exclusion chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry analysis were employed for peptide purification and identification. Finally, two peptides were identified, both with linear structures, consisting of amino acid sequences TCGGQGR and KEAGAFIDR. At 1 mg/mL, the heptapeptide performed better than the nonapeptide in terms of wheat growth and health, but neither peptide exhibited antimicrobial activity. Only the heptapeptide displayed significant antioxidant activity, and this activity bioaccumulated in wheat leaves after 7 days of hydroponic growth. The heptapeptide did not match any known metabolites in PepBank, BIOPEP, UniProt or METLIN databases, and is therefore a novel peptide with potential as a functional fertiliser component.

Sesame water-soluble proteins fraction contains endopeptidases and exopeptidases with high activity: A natural source for plant proteases

Recently, the interest in the plant proteases has greatly increased. However, only a few of proteases are isolated from the hugely produced oilseeds for the practical utilizations. In this study, the raw sesame milk prepared from peeled sesame seeds was separated into floating, skim, and precipitate fractions by centrifugation. The predominant aspartic endopeptidases and serine carboxypeptidases, which exerted high synergetic activity at pH 4.5–5 and 50–60 °C, were identified in the skim by the liquid chromatography tandem mass spectrometry, Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis, protease inhibitor assay, trichloroacetic acid–nitrogen soluble index (TCA–NSI), and free amino acid analyses. By incubating the mixture (protein content, 2%) of skim and precipitate at pH 4.5 and 50 °C for 6 h, the TCA–NSI and free amino acids achieved to 38.42% and 3148 mg/L, respectively. Moreover, these proteases efficiently degraded the proteins from soybean, peanut, and bovine milk.

Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophilamelanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.

Effect of Hydrophobicity on the Anticancer Activity of Fatty-Acyl-Conjugated CM4 in Breast Cancer Cells.

Antimicrobial peptides (AMPs) are important anticancer resources, and exploring AMP conjugates as highly effective and selective anticancer agents would represent new progress in cancer treatment. In this study, we synthesized C4–C16 fatty-acyl-conjugated AMP CM4 and investigated its physiochemical properties and cytotoxicity activity in breast cancer cells. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) showed that long-chain fatty acyl (≥C12) conjugation prevented N-acyl-CM4 from trypsin hydrolysis. RP-HPLC and circular dichroism (CD) spectra showed that the hydrophobicity and helical content of N-acyl-CM4 increased with the acyl length. The acyl chain length was positively related to the cytotoxicity of C8–C16 conjugates, and C12–C16 fatty acyl conjugates exhibited significant cytotoxicity against MX-1, MCF-7, and MDA-MB-231 cells, with IC50 values <8 μM. Flow cytometry and confocal laser scanning microscopy results showed that N-acylated conjugation significantly increased the membrane affinity in breast cancer cells, and C12–C16 acyl conjugates were capable of translocating to the intracellular space, thereby targeting mitochondria and inducing apoptosis. N-acyl-CM4 showed low cytotoxicity against normal mammalian cells and erythrocytes, especially ≤C12 fatty acyl conjugates, exhibiting selective cytotoxicity to breast cancer cells. The current work indicated that increasing hydrophobicity by attaching long fatty acyl (≥C12) to AMPs may be an effective method to improve the anticancer activity, together with selectivity and resistance to trypsin hydrolysis. This finding provides a good strategy to develop AMPs as effective anticancer agents in the future.

Purification of Nucleoprotein of H9N2 Avian Influenza Virus Strain by Electroelution

Background: Avian influenza virus (AIV) belongs to the family of Orthomyxoviruses type A and causes avian influenza (AI) infectious disease. Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzyme-linked immunosorbent assay (ELISA) are considered as important tools for the antibodies detection against viral antigens. Due to antigenic variation in the surface of AIV glycoproteins (hemagglutinin and neuraminidase), these proteins cannot be used in serological tests. Development of assays to detect AI surface glycoproteins is problematic because a great variety of combinations of these subtypes are found in nature. The internal antigen determinants on the nucleoprotein (NP) are highly conserved within influenza viruses, making this protein more appropriate for a serological test. Objectives: In the experimental present study, an effectual method was expanded to purify NP of H9N2 AIV based on Electroelution method. Methods: AIV strain A/flash chicken/Iran/772/1998 (H9N2) was acquired from the Department of Avian Influenza Reference Laboratory, Razi Vaccine and Serum Research Institute, Iran, about 2 cc, in 2017. Nucleoprotein of AIV (H9N2) was purified by an efficient and simple modified method directly from native polyacrylamide gel electrophoresis (PAGE) according to the Electroelution method. The purified protein concentration was defined by the Lowry method, and the purified NP protein (60 KDa) was examined by Tricine-SDS-PAGE. Results: The protein concentration of the virus solution was 4.62 mg/mL by the Lowry method. The purified Nucleoprotein concentration was 0.296 mg/mL by Lowry method and the Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed only a 60-KDa protein band in the gel. Conclusions: The current technique was simple and rapid and made it possible to isolate NP from the H9N2 virus. The Nucleoprotein antigen is an appropriate candidate potential to detect antibodies against all subtypes of AIV and used as the main target antigen for the diagnosis of influenza virus due to its very high scale of sequence preserved among exist strains.

The wound healing potential of collagen peptides derived from the jellyfish Rhopilema esculentum

PurposeWound represents a major health challenge as they consume a large amount of healthcare resources to improve patient's quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration. MethodsIn this study, collagen was extracted from the jellyfish--Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software. ResultsThe yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP1) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP2). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 μg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in β-fibroblast growth factor (β-FGF) and the transforming growth factor-β1 (TGF-β1) expression on collagen peptides treated group. ConclusionCollagen peptides derived from the jellyfish–Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.

Tricine-SDS-PAGE.

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) is an efficient way of separating low molecular mass proteins. However, the standard system is quite complicated and specifically may not be useful when the separated proteins are to be recovered from the gel for quantitative analysis. Here, we describe a simplified system whereby these smaller proteins can be resolved in comparatively low percentage gels which have high compatibility with modern detectors such as UV and inductively coupled plasma-mass spectrometry (ICP-MS).

Acid whey proteolysis to produce angiotensin-I converting enzyme inhibitory hydrolyzate

α-Lactalbumin (ALA) and β-lactoglobulin (BLG) are the main proteins in acid whey and proteolytic digestion of these proteins provides a source of peptides that inhibit angiotensin-I converting enzyme (ACE-I). Enzymatic hydrolysis of acid whey proteins using cysteine peptidase enzyme extract (antiacanthain) from ripe Bromelia antiacantha Bertol fruit was investigated. The effect of pH on hydrolysis kinetics of ALA and BLG by antiacanthain was analysed at 50 °C. Antiacanthain had a high catalytic efficiency for ALA (1.3 × 104 M−1 s−1) and BLG (1.7 × 104 M−1 s−1) at pH 9.2. The effect of temperature and amount of antiancanthain (enzyme to substrate ratio or E/S) on acid whey hydrolysis were studied by measurement of degree of hydrolysis (DH) and protein degradation profile evaluated by tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine–SDS-PAGE). Optimal acid whey hydrolysis was at pH 9.2, E/S = 1.0 (U/mg) and 50 °C. BLG was degraded faster than that of ALA, but after 30 min hydrolysis almost all BLG and ALA were degraded and new bands corresponding to peptides with a mass lower than 3.0 kDa appeared. Fractions of acid whey (DH = 12.3%), ALA (DH = 17.3%) and BLG (DH = 30.6%) hydrolyzates showed ACE-I inhibitory activity. Results also suggest that most inhibitory activity might be due to low molecular weight peptides from ALA, since 3 kDa permeate of ALA hydrolyzate showed the highest IC50 value (1.7 ± 0.4 µg/mL). Interestingly, low molecular weight peptides from both ALA and BLG had low immunoreactivity levels, monitored by latex agglutination tests, suggesting that these peptides could be safe for use by those with strong allergic responses. Results indicate that antiacanthain can be used as an enzymatic preparation for acid whey hydrolysis. The potential use of this hydrolyzate for manufacture of health-promoting products of high added value is discussed.

Isolation, Characterization and Amino Acid Composition of a Bacteriocin Produced by Bacillus methylotrophicus Strain BM47.

SUMMARYMembers of the bacterial genus Bacillus are known as producers of a broad spectrum of antibiotic compounds of proteinaceous nature that possess inhibitory activity against different saprophytic and pathogenic microorganisms. In the current research, a peptide synthesized by Bacillus methylotrophicus strain BM47, previously isolated from a natural thermal spring in Bulgaria, was identified and characterized as a bacteriocin. In vitro antimicrobial screening of the crude bacteriocin substance of B. methylotrophicus BM47 showed activity against the plant pathogenic fungi Fusarium moniliforme, Aspergillus awamori, Penicillium sp., Aspergillus niger and Gram-negative bacterium Pseudomonas aeruginosa. The antimicrobial activity of the crude bacteriocin substance was partially inhibited by the enzymes trypsin, Alcalase®, Savinase®, proteinase K, papain and Esperase®, while catalase was not effective. The crude bacteriocin substance was relatively pH resistant, but sensitive to the action of heat and most organic solvents and detergents tested. To obtain the active protein fractions, crude bacteriocin substance was purified by fast protein liquid chromatography (FPLC) using a strong anion exchange column. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purified bacteriocin had molecular mass of 19 578 Da. The amino acid analysis performed by high-performance liquid chromatography (HPLC) revealed that the isolated bacteriocin consisted of 17 types of amino acids, with the highest mol fraction expressed as percent of serine (29.3), valine (10.3), alanine (9.8) and tyrosine (7.1).

Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA.

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.

Immunochemical Identification of Immunoreactive Ghrelin in Tea Plant, Camellia sinensis (L) O. Kuntze

Ghrelin was originally described as a growth hormone (GH)-releasing and appetite stimulating hormone in mammals, which is highly conserved and extensively expressed in all eukaryotic organisms investigated. Ghrelin is a natural ligand of the growth hormone secretagogue-receptor, and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Although ghrelin has been well characterized in mammalian systems, the most recent investigations demonstrated that plants have ghrelin immunoreactivity. The aim in this study is to determine ghrelin-like immunoreactive substances in fresh vegetative tissue of tea plant [Camellia sinensis (L.) O. Kuntze (Theaceae)]. Total protein was extracted from the plant samples by using trichloroacetic acid/acetone precipitation. Ghrelin-like immunoreactive substances in the tea plant extracts were investigated with indirect enzyme-linked immunosorbent assay method. From the tea plant samples human-ghrelin-like immunoreactive substances were isolated and characterized using tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting techniques. The ghrelin immunoreactivity of tea extract and human serum were found to be 1.94 ± 0.27 and 0.31 ± 0.012 ng/mg protein, respectively. As a result, in fresh vegetative tissues of tea a single ghrelin-like protein band with a molecular mass of ~13 kDa was observed using tricine-SDS-PAGE and western blotting. This study reflects the importance of ghrelin in living organisms, and speculates the ghrelin-like bioactive peptides derived from plants may play a role in the regulation of appetite and body weight control.

Effects of Disulfide Bond Reduction on the Conformation and Trypsin/Chymotrypsin Inhibitor Activity of Soybean Bowman-Birk Inhibitor.

Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors (BBIs). In this study, highly purified BBI-A (approximately 96%) was obtained from soybean whey at the 0.1 g level by the complex coacervation method. BBI-A has seven disulfide bonds (SS) and no sulfhydryl group and exhibits trypsin inhibitor activity (TIA) and chymotrypsin inhibitor activity (CIA). The X-ray structure has shown that BBI-A has five exposed SS and two buried SS. Because of steric hindrance, it was reasonable to consider that dithiothreitol first attacks the five exposed SS and then the two buried SS, which was supported by the results that SS reduction with dithiothreitol could be divided into quick and slow stages, and the critical point was close to 5/7. The effects of SS reduction on TIA and CIA could be divided into three stages: when one exposed SS was reduced, both TIA and CIA decreased to approximately 60%; with further reduction of exposed SS, CIA gradually decreased to 8% and TIA gradually decreased to 26%; with further reduction of buried SS, CIA gradually decreased to 2% and TIA slightly decreased to 24%. Far-ultraviolet (far-UV) circular dichroism (CD) spectra showed that the secondary structure of BBI-A was slightly changed, whereas near-ultraviolet (near-UV) CD spectra showed that the conformation of BBI-A was substantially changed after the five exposed SS were reduced; further reduction of buried SS affected the conformation to some extent. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from a C8 column showed the same trend as near-UV CD spectra. BBI-A has a structural peculiarity in that two hydrophobic patches are exposed to the exterior (in contrast to typical soluble proteins), which was attributed to the seven SS by some researchers. These results support the hypothesis that hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core occurred after the reduction of SS in BBI-A.

Use of Different Proteases to Obtain Flaxseed Protein Hydrolysates with Antioxidant Activity.

The antioxidant activity of flaxseed protein hydrolysates obtained using five different enzymes was evaluated. Proteins were isolated from flaxseed cake and were separately treated with papain, trypsin, pancreatin, Alcalase and Flavourzyme. The degree of hydrolysis (DH) was determined as the percentage of cleaved peptide bonds using a spectrophotometric method with o-phthaldialdehyde. The distribution of the molecular weights (MW) of the hydrolysis products was profiled using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC) separations. The antioxidant activities of the protein isolate and hydrolysates were probed for their radical scavenging activity using 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•+) and photochemiluminescence (PCL-ACL) assays, and for their ferric reducing antioxidant power (FRAP) and ability to bind Fe2+. The hydrolysates were more effective as antioxidants than the protein isolate in all systems. The PCL-ACL values of the hydrolysates ranged from 7.2 to 35.7 μmol Trolox/g. Both the FRAP and ABTS•+ scavenging activity differed among the hydrolysates to a lower extent, with the ranges of 0.20–0.24 mmol Fe2+/g and 0.17–0.22 mmol Trolox/g, respectively. The highest chelating activity (71.5%) was noted for the pancreatin hydrolysate. In general, the hydrolysates obtained using Alcalase and pancreatin had the highest antioxidant activity, even though their DH (15.4% and 29.3%, respectively) and the MW profiles of the peptides varied substantially. The O2•− scavenging activity and the ability to chelate Fe2+ of the Flavourzyme hydrolysate were lower than those of the Alcalase and pancreatin hydrolysates. Papain was the least effective in releasing the peptides with antioxidant activity. The study showed that the type of enzyme used for flaxseed protein hydrolysis determines the antioxidant activity of the hydrolysates.

Gastric protein hydrolysis of raw and roasted almonds in the growing pig

Gastric protein hydrolysis may influence gastric emptying rate and subsequent protein digestibility in the small intestine. This study examined the gastric hydrolysis of dietary protein from raw and roasted almonds in the growing pig as a model for the adult human. The gastric hydrolysis of almond proteins was quantified by performing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent image analysis. There was an interaction between digestion time, stomach region, and almond type for gastric protein hydrolysis (p<0.05). Gastric emptying rate of protein was a significant (p<0.05) covariate in the gastric protein hydrolysis. In general, greater gastric protein hydrolysis was observed in raw almonds (compared to roasted almonds), hypothesized to be related to structural changes in almond proteins during roasting. Greater gastric protein hydrolysis was observed in the distal stomach (compared to the proximal stomach), likely related to the lower pH in the distal stomach.

Antioxidant Activity of Enzymatic Hydrolysate Derived From Hairtail Surimi Wash Water Using an Immobilized Chymotrypsin-Trypsin Column Reactor

In this study, a column reactor filled with immobilized chymotrypsin–trypsin (I-CT) was used to prepare antioxidant peptides from hairtail surimi wash water. At the optimum reaction temperature (30C), when residence time is 5 min (flow rate is 3 mL/min), the conversion rate of the I-CT column enzymatic reactor could be maintained above 60%. The half-life of the column reactor was in excess of 6 h. At 30C, when residence time is 15 min (flow rate is 1 mL/min), the antioxidant capacity of the resulting enzymatic hydrolysate was the highest (1.59 mmol/L FeSO4 equivalent) and the product conversion rate was 67.0%. Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed that the main ingredients of enzymatic hydrolysate were low-molecular-weight peptides (<4 kDa). Practical Applications In order to fully utilized protein resources, it is necessary to develop an effective approach to recover protein in the surimi wash water. To this end, a practical method by using a column reactor filled with immobilized chymotrypsin–trypsin developed in our laboratory was established. The hydrolysates obtained by this method may be used as ingredients of functional foods due to their antioxidant activity.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Molecular Cloning of Hemocyanin cDNA from Fenneropenaeus chinensis and Antimicrobial Analysis of Two C-terminal Fragments

Peptides derived from shrimp hemocyanin have antimicrobial properties. This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments. Based on sequence analysis of Fenneropenaeus chinensis hemocyanin FCHc, we subcloned two FCHc fragments by designing special primers. Two antimicrobial peptides (AMPs) were derived from FCHc (FCHc-C1 and FCHc-C2). The recombinant sequence of FCHc-C1 consisted of 207bp encoding 69 amino acids and the recombinant sequence of FCHc-C2 consisted of 120bp encoding 40 amino acids. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that recombinant FCHc-C1 and FCHc-C2 peptides (rFCHc-C1 and rFCHc-C2) were expressed successfully. An inhibition assay showed that FCHc-C1 and FCHc-C2 were anionic AMPs with antifungal and antibacterial activities.

Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site

PEGylation has been a successful strategy for improving the pharmacokinetic and pharmaceutical properties of proteins and peptides. However, PEGylated products also create significant challenges for detailed structural characterization. In this work, a site-specific PEGylation strategy was successfully performed on an exendin-4 analog (Ex4C) through a maleimide method. Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), analytical reversed phase HPLC (RP-HPLC) and MALDI-TOF were applied to verify the accomplishment of the PEGylation. Peptide mapping was investigated after tryptic digestion, and the PEGylaton site was successfully located on the C-terminal fragment of Ex4C. Amino acid analysis (AAA) of cysteine was then applied to verify the block in the thiol group caused by PEGylation. We believe that the combination of proper enzymatic digestion and amino acid analysis of cysteine provided an easy and convincing way to identify the PEGylation site in this maleimide method.

Immobilization and Characterization of a Thermostable Lipase

Lipases have found a number of commercial applications. However, thermostable lipase immobilized on nanoparticle is not extensively characterized. In this study, a recombinant thermostable lipase (designated as TtL) from Thermus thermophilus WL was expressed in Escherichia coli and immobilized onto 3-APTES-modified Fe3O4@SiO2 supermagnetic nanoparticles. Based on analyses with tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer observation, the diameter of immobilized lipase nanoparticle was 18.4 (± 2.4) nm, and its saturation magnetization value was 52.3 emu/g. The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field. The biochemical characterizations revealed that, comparing with the free one, the immobilized lipase exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The K m value for the immobilized TtL (2.56 mg/mL) was found to be lower than that of the free one (3.74 mg/mL), showing that the immobilization improved the affinity of lipase for its substrate. In addition, the immobilized TtL exhibited good reusability. It retained more than 79.5 % of its initial activity after reusing for 10 cycles. Therefore, our study presented that the possibility of the efficient reuse of the thermostable lipase immobilized on supermagnetic nanoparticles made it attractive from the viewpoint of practical application.