Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively.
In this study, the methods for extraction and purification of miraculin from Synsepalum dulcificum were investigated. For extraction, the effect of different extraction buffers (phosphate buffer saline, Tris-HCl and NaCl) on the extraction efficiency of total protein was evaluated. Immobilized metal ion affinity chromatography (IMAC) with nickel-NTA was used for the purification of the extracted protein, where the influence of binding buffer pH, crude extract pH and imidazole concentration in elution buffer upon the purification performance was explored. The total amount of protein extracted from miracle fruit was found to be 4 times higher using 0.5M NaCl as compared to Tris-HCl and phosphate buffer saline. On the other hand, the use of Tris-HCl as binding buffer gave higher purification performance than sodium phosphate and citrate-phosphate buffers in IMAC system. The optimum purification condition of miraculin using IMAC was achieved with crude extract at pH 7, Tris-HCl binding buffer at pH 7 and the use of 300 mM imidazole as elution buffer, which gave the overall yield of 80.3% and purity of 97.5%. IMAC with nickel-NTA was successfully used as a single step process for the purification of miraculin from crude extract of S. dulcificum.
To compare the specific intracellular proteinase A activity in clinical isolates of Candida species isolated from Iranian and Malaysian patients, the blood and kidneys of mice infected by Candida cells isolated from these human patients.
Adsorption of lysozyme on the dye-affinity nanofiber membranes was investigated in batch and dynamic modes. The membrane matrix was made of electrospun polyacrylonitrile nanofibers that were grafted with ethylene diamine (EDA) and/or chitosan (CS) for the coupling of Reactive Blue 49 dye. The physicochemical properties of these dye-immobilized nanofiber membranes (P-EDA-Dye and P-CS-Dye) were characterized microscopically, spectroscopically and thermogravimetrically. The capacities of lysozyme adsorption by the dye-affinity nanofiber membranes were evaluated under various conditions, namely pH, dye immobilized density, and loading flow rate. The adsorption of lysozyme to the dye-affinity nanofiber membranes was well fitted by Langmuir isotherm and pseudo-second kinetic models. P-CS-Dye nanofiber membrane had a better performance in the dynamic adsorption of lysozyme from complex chicken egg white solution. It was observed that after five cycles of adsorption-desorption, the dye-affinity nanofiber membrane did not show a significant loss in its capacity for lysozyme adsorption. The robustness as well as high dynamic adsorption capability of P-CS-Dye nanofiber membrane are promising for the efficient recovery of lysozyme from complex feedstock via nanofiber membrane chromatography.
Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.
Dye-ligand affinity chromatography in a stirred fluidized bed has been developed for the rapid recovery of malate dehydrogenase (MDH) from highly turbid baker's yeast cell homogenate in a single step. The most suitable dye, namely Reactive Orange 4, in its optimal immobilized concentration of 8.78 mg/mL was immobilized onto high-density STREAMLINE matrix. To further examine optimal adsorption and elution conditions, the enzyme recovery operation was carried out using unclarified cell homogenates in stirred fluidized bed system. Aiming to develop a non-specific eluent, namely NaCl, to effectively elute the MDH adsorbed, direct recovery of MDH from highly turbid cell homogenate (50% w/v) in a stirred fluidized bed adsorption system was performed. The proposed system successfully achieved a recovery yield of 73.6% and a purification factor of 73.5 in a single step by using 0.6 M NaCl as an eluent at a high liquid velocity of 200 cm/h.
Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
The prevalence of oral squamous cell carcinoma (OSCC) is high in South and Southeast Asia regions. Most OSCC patients are detected at advanced stages low 5-year survival rates. Aberrant expression of glycosylated proteins was found to be associated with malignant transformation and cancer progression. Hence, identification of cancer-associated glycoproteins could be used as potential biomarkers that are beneficial for diagnosis or clinical management of patients. This study aims to identify the differentially expressed glycoproteins using lectin-based glycoproteomics approaches. Serum samples of 40 patients with OSCC, 10 patients with oral potentially malignant disorder (OPMD), and 10 healthy individuals as control group were subjected to two-dimensional gel electrophoresis (2-DE) coupled with lectin Concanavalin A and Jacalin that specifically bind to N- and O-glycosylated proteins, respectively. Five differentially expressed N- and O-glycoproteins with various potential glycosylation sites were identified, namely N-glycosylated α1-antitrypsin (AAT), α2-HS-glycoprotein (AHSG), apolipoprotein A-I (APOA1), and haptoglobin (HP); as well as O-glycosylated AHSG and clusterin (CLU). Among them, AAT and APOA1 were further validated using enzyme-linked immunosorbent assay (ELISA) (n = 120). It was found that AAT and APOA1 are significantly upregulated in OSCC and these glycoproteins are independent risk factors of OSCC. The clinical utility of AAT and APOA1 as potential biomarkers of OSCC is needed for further evaluation.
Monoclonal antibodies (mAbs) are unique and specific drug molecules targeting the treatment of various diseases such as arthritis, immune disorders, infectious diseases, and cancer etc. Different methods such as antibody coupled affinity chromatography, hydrophobic interaction chromatography, etc., can be applied to purify mAbs from various sources. This article provides a simple, cost effective, preparative native-polyacrylamide gel electrophoresis (n-PAGE)technique to purify mAbs expressed in H-192 cells (Hybridoma murine cell lines) against an antigen i.e. 17-alpha-hydroxyprogesterone (17-OHP), which further can have diagnostic application to detect Congenital Adrenal Hyperplasia (CAH). Furthermore, different parameters such as concentration and volume of the feedstock (medium containing antibodies), pore size of gel, height of resolving gel etc. were optimized to obtain the maximum purity and yield of mAbs.
There are numerous reports on poly-β-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.
The use of lectin affinity chromatography prior to 2-DE separation forms an alternative method to unmask the expression of targeted glycoproteins of lower abundance in serum samples. Reduced expression of alpha-2 macroglobulin (AMG) and complement factor B (CFB) was detected in sera of patients with nasopharyngeal carcinoma (NPC) when pooled serum samples of the patients and those of healthy individuals were subjected to affinity isolation using immobilized champedak mannose-binding lectin and analyzed by 2-DE and densitometry. The AMG and CFB spots were not detected in the 2-DE protein profiles when the same pooled serum samples were subjected to albumin and IgG depletion and neither were they detected when the depleted samples were analyzed by western blotting and lectin detection. Together with other acute-phase response proteins that were previously reported to be altered in expression in NPC patients, AMG and CFB may serve as useful complementary biomarkers for NPC.
Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1-252 and 253-371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.
Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.
Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata. HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions.
GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
We recently adopted immobilized jacalin as an affinity adsorbent to purify human serum IgA for laboratory study. In the course of our investigation, we detected a serum protein that co-eluted with IgA from jacalin-agarose affinity column. It constituted in significant quantity (24.0 +/- 0.9%, n = 30) of total jacalin-bound protein (JBP) and the yield was equivalent to 0.4 +/- 0.1 mg per ml serum. The molecular mass of this protein was 55 kDa with electromobility in the alpha 2 region as demonstrated by SDS-PAGE and immunoelectrophoresis. N-terminal microsequencing of this 55 kDa protein revealed that it is human alpha 2-HS glycoprotein (alpha 2HSG). The molecular interaction of alpha 2HSG with jacalin was characterized by competitive ELISA: human serum IgA, human colostrum secretory IgA (sIgA), and monosaccharides including D-galactose and melibiose exhibited strong inhibitory effect on its binding to jacalin. Accordingly, we propose that human alpha 2HSG binds in a similar manner as that of the bovine fetuin to jacalin. In addition, alpha 2HSG displays similar binding property to jacalin from different geographic area (India and Malaysia) and from different laboratory preparations (Sigma, Pierce and 'homemade' jacalin).
A confirmatory and quantitative HPLC-tandem mass spectrometry (MS-MS) method for human chorionic gonadotropin hormone (hCG) at concentrations as low as 5 IU/l following immunoaffinity extraction of the glycoprotein from urine was developed. The extraction method involved retention of urinary hCG in the immunoaffinity column via specific antigen-antibody interaction. A variety of eluents were then used to quantitatively elute hCG from the immunoaffinity column. Qualitative and quantitative analysis of hCG were undertaken using MS-MS by identifying the amino acid sequence of the marker peptide betaT5 obtained from hCG by tryptic digestion and the peak areas of three product ions b(6)(+), b(9)(+) and y(11)(+), respectively.
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.
Pseudobranch function has long interested scientists, but its role has yet to be elucidated. Several studies have suggested that pseudobranchs serve respiratory, osmoregulatory, and sensory functions. This work investigated the immunolocalization of pseudobranch carbonic anhydrase (CA) in the teleost fish species rainbow trout (Oncorhynchus mykiss) to clarify its physiological function. CA was purified from rainbow trout gills O. mykiss and specific antibodies were raised. Immunoblotting between tissue homogenates of pseudobranch and gill CA antibodies showed specific immunostaining with only one band corresponding to CA in the pseudobranch homogenate. Results of immunohistochemical technique revealed that CA was distributed within pseudobranch cells and more precisely in the apical parts (anti-vascular) of cells. The basal (vascular) parts of cells, tubular system, blood capillaries, and pillar cells were not immunostained. Immunocytochemistry confirmed these results and showed that some CA enzyme was cytoplasmic and the remainder was linked to membranous structures. The results also showed that the lacunar tissue layers did not display immunoperoxidase activity. Our results indicated that pseudobranch CA may have a function related to the extracellular medium wherein CA intervenes with the mechanism of stimulation of afferent nerve fibers.