A simple scheme for large scale purification of urine - Derived Bence Jones Kappa protein.

We have investigated and optimized purification process, suitable for industrial scale, to obtain high purity grade Bence Jones Kappa Protein ('BJK-Protein'), while preserving its physiological properties and functions. BJK-Protein was obtained from a biological waste product i.e. human urine of renal failure patients. Isolated 'BJK-Protein' was analyzed by electrophoresis, western blotting, double immunodiffusion, SEC-HPLC assay and Mass Spectrometry (MS). The relative molecular mass of 'BJK-Protein' is 23054.2 Da. Moreover, dimer forms of 'BJK-Protein' were also detected in SDS-PAGE and mass spectrum corresponding to 46054.4 Da. The results of western blotting, immunoelectrophoresis, SEC-HPLC assay, and mass spectrometry analysis indicate a high purity (>99 %) of 'BJK-Protein'. Peptide mass fingerprint analysis of 'BJK-Protein' yielded peptides that partially matches the known database sequences of kappa variable region (KV139_HUMAN) of immunoglobulin. This protein was found to be stable up to 20 months at 2-8 °C temperature and also found negative for major undesirable viral markers as well as bacterial endotoxin. With this purification approach, the cost of purified 'BJK-Protein' is significantly reduced as compared to the current market price of Kappa light chain available in international market.

An improved protocol for large scale production of high purity 'Fc' fragment of human immunoglobulin G (IgG-Fc).

We describe a simplified approach for purification and characterization of human 'IgG-Fc' fragment used widely as immunochemical tool and for therapeutic purposes. The 'Fc' fragment was purified from human IgG in a 3-stage column chromatography. The purified 'Fc' fragment appeared as a dimer glycoprotein with an apparent molecular mass of 52,981 Dalton (Ultraflex MALDI TOF/TOF). The Size-exclusion HPLC profile of the purified 'Fc' fragment of human IgG matched that of a commercially procured reference 'Fc' fragment material. The purity of the 'Fc' fragments was >99% by SDS-PAGE and size-exclusion HPLC. The results of Western blotting, immunoelectrophoresis, and mass spectrometry analysis indicate a high purity of the 'Fc' fragment. Peptide mass fingerprint analysis of the purified 'Fc' protein yielded peptides that partially match the known database sequences of FCG3B_HUMAN (Uniprot ID: O75015). This method of purification of the 'Fc' fragment is suitable for achieving high purity level of 'Fc' fragment protein. With this purification approach, the cost of the purified 'Fc' fragment of human IgG is significantly reduced as compared with the current market price of IgG-Fc fragment protein in international market. The purified 'IgG-Fc' fragment protein was found to be negative for major viral markers.

Vaccine development against coronavirus (2003 to present): An overview, recent advances, current scenario, opportunities and challenges.

BACKGROUND AND AIM: The pandemic COVID-19 occurring due to novel emerging coronavirus-2019 (SARS-CoV-2) is severely affecting the worldwide public health, culture, economy and human social behaviour. Till date, there is no approved medicine/treatment to cure COVID-19, whereas, vaccine development efforts are going on high priority. This review aimed to provide an overview of prior art, recent advances, vaccine designing strategies, current scenario, opportunities and challenges related to development of coronavirus vaccine. METHOD: A literature survey was conducted using Scopus, PubMed and Google Scholar with the search key as: coronavirus vaccine, SARS vaccine, MERS vaccine and COVID-19 vaccine. Articles related to above search query were retrieved, sorted, analyzed and developed into an easy-to-understand review. RESULTS: The genome phylogenetic analysis suggested that genomic sequence of SARS-CoV-2 is almost 80% similar to that of SARS-CoV, further both these viruses bind to same host cell receptor ACE-2. Hence it is expected that, previously available literature data about coronavirus vaccine designing may play crucial role in development of rapid vaccine against COVID-19. In view of this, the present review discuss (i) existing information (from 2003 to present) about the type of vaccine, antigen, immunogenic response, animal model, route of administration, adjuvants and current scenario for designing of coronavirus vaccine (ii) potential factors and challenges related to rapid development of COVID-19 vaccine. CONCLUSION: In conclusion, we discuss possible clues/ target sites for designing of vaccine against SARS-CoV-2 virus based on prior-art.

A cost-effective method for purification and characterization of human urinary albumin.

We describe a simplified approach for the purification and characterization of urinary albumin, a key biomarker currently used for understanding the onset and prognosis of microalbuminuria. Urinary albumin was purified from human urine collected from diabetic kidney disease patients by using 2-stage tangential flow filtration process and set of column chromatography steps. The relative molecular mass of urinary albumin is 66,871 Da (SYNAPT G2 High Definition Mass Spectrometry System). Isolated urinary albumin was analyzed by SDS-PAGE, Western blotting, immunoelectrophoresis, Ouchterlony double-immunodiffusion, single radial immunodiffusion, size-exclusion HPLC and peptide mass fingerprint analysis. The size-exclusion HPLC elution profile of the purified urinary albumin was similar to that of a reference form of native albumin. Peptide mass fingerprint analysis of the purified urinary albumin yielded peptides that partially matched with known sequence of ALBU_HUMAN (P02768). This is the first report of purification and validation of immunochemically reactive form of urinary albumin from a large volume of urine of diabetic kidney disease patients. In this purification approach, the cost of the purified albumin is significantly lower.

Purification of recombinant human thyroid peroxidase (hTPO) from AD293 mammalian cells.

Human thyroid peroxidase (hTPO) has been secretory expressed in AD293 mammalian cells. cDNA sequence of 'Gluc' (Gaussia luciferase) protein from Gaussia princeps was incorporated at the amino terminal of hTPO gene for secretion of targeted protein outside the mammalian cells. Augmentation of TPO clone in serum free mediums was investigated and a simplified purification procedure of hTPO has been reported here. Purified hTPO was further analyzed by SDS-PAGE and immunoblotting (western blotting). The relative molecular mass of hTPO was found to be 105kDa. This is the first report with respect to cost effective and simplified purification approach to get highest yield and purity of recombinant hTPO.

Simplified approach for in-vitro production and purification of cell derived Cancer Antigen 15-3.

Cancer antigen 15-3 (CA15-3) is a key biomarker, currently used for understanding the onset and prognosis of breast cancer. In present investigation, CA15-3 has been purified from the culture supernatant of breast cancer T47-D cell line with 76% yield and 3350 fold purification. Isolated CA15-3 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting (western blotting), chemiluminescence immunoassay (CLIA) and Fourier-transform infrared spectroscopy (FTIR). CA15-3 is a monomeric protein with an apparent molecular mass in between ∼250-350kDa. The FTIR spectroscopy revealed similar profiles of T47-D derived CA15-3 and commercially available CA15-3 protein. With the easy availability of T47-D cell line and a simple purification approach described here will support for the large scale production of CA15-3 to be used for various clinical and diagnostic applications.

Simplified purification approach of urinary neutrophil gelatinase-associated lipocalin by tangential flow filtration and ion exchange chromatography.

This investigation reports a simplified approach for the purification of urinary siderocalin known as neutrophil gelatinase-associated lipocalin (NGAL). Urinary NGAL was purified by tangential flow filtration and ion exchange chromatography. Isolated NGAL was analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular mass of NGAL is 23674Da. Peptide mass fingerprinting of the purified NGAL yielded peptides that partially matched with known sequence of P80188 (NGAL_HUMAN). The tryptic digestion profile of isolated NGAL infers that it may be unique and additive molecule in the dictionary of urinary proteins. This is the first report of purification and validation of urinary NGAL from large volume sample by using tangential flow filtration and peptide sequencing respectively. This cost-effective and simplified approach to purification of NGAL, together with the easy availability of urine sample makes the large-scale production of NGAL possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

Development of an indirect immunofluorescence based assay for diagnosis of ulcerative colitis in Indian population.

The prevalence of Ulcerative Colitis (UC), once thought to be negligible, has increases exponentially in the Indian population. The development of novel, cost effective and time efficient Indirect Immunofluorescence (IIF) based assay for detection of anti-neutrophil cytoplasmic antibodies (ANCA) and diagnosis of UC in the Indian population is discussed. A novel IIF based assay was developed using intact nuclei from human neutrophils to detect atypical p-ANCA in patients suffering from UC. Sera from 45 patients diagnosed with UC, 45 healthy controls and one related disease control were tested using a novel UC-ANCA assay and validated by commercially available ANCA IIF assay. Prevalence of ANCA amongst UC patients in the Indian population was determined. Atypical p-ANCA was detected in 86.6% of the patients using the UC-ANCA assay as compared to 71.1% using the commercial ANCA assay. The validation of UC-ANCA assay with a commercially available ANCA IIF assay resulted in higher sensitivity. The UC-ANCA assay proved to be not only enhanced in terms of performance but also comparatively economical and rapid. The novel UC-ANCA assay may prove to be very useful in identification and differentiation of UC patients from typical ANCA positive subjects suffering from other autoimmune diseases at one tenth the cost of clinically available ANCA IIF tests which will immensely benefit the cost constrained diagnostic field of developing countries.

A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

Development of glycan specific lectin based immunoassay for detection of prostate specific antigen.

We describe an analytical approach for the detection and verification of glycosylation patterns of prostate specific antigen (PSA), a key biomarker currently used for understanding the onset and prognosis of prostate cancer. PSA has been purified from the human seminal plasma and total PSA from prostate cancer sera. PSA is a monomeric glycoprotein with an apparent molecular mass 28040.467 Da, which exhibits a characteristic protease activity against casein and gelatin. Its optimal protease activity is centered on neutral pH. Peptide mass fingerprint analysis of the purified PSA has yielded peptides that partially match with known database sequences (Uniprot ID P07288). Tryptic digestion profile of isolated PSA, infer the exclusive nature of PSA and may be additive molecule in the dictionary of seminal proteins. Surface plasmon resonance and lectin immunoassay revealed direct interaction between a newly developed anti-PSA monoclonal antibody (C4E6) and PSA. A lectin based immunoassay is reported here which was achieved with the C4E6 anti-PSA antibody and biotinylated plant lectins. This investigation provides an alternative method to isolate and quantify PSA with altered glycosylation which might be seen in the prostate cancer and developing a lectin based immunoassay to detect PSA in serum of prostate cancer patients.

Evaluation of a lactogenic activity of an aqueous extract of Cyperus rotundus Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: In Ayurveda, the rhizome of Cyperus rotundus Linn has been reported for wide spectrum of biological activities including lactational therapy for increasing milk quantity. However, not a single report is available on validation of its herbal galactagogue potentiality in literature. Thus, the present study is aimed to assess the lactogenic property of aqueous extract of Cyperus rotundus (CRE). MATERIALS AND METHODS: The effect of aqueous extract of Cyperus rotundus rhizome was evaluated by measuring weight of the pups during suckling period. Quantitatively, total protein and carbohydrate contents of mammary tissue and serum prolactin and cortisol level were calculated. Histopathological analysis of mammary gland, pituitary gland, heart, liver, spleen, kidney, and ovary tissues was carried out. Acute toxicity of CRE against rat was assessed by the Hippocratic test and biochemical profile of blood serum. RESULTS: Oral administration of 300 and 600mg of CRE induced about 23% and 40% more milk in experimental group of animals as compared to the control group of animals. Weight gain by pups and mother rats of treated groups were significantly higher following administration of CRE as compared to that of control group. Moreover protein and carbohydrate content of mammary gland tissue were also significantly more than control group of animals. The CRE was found to stimulate the synthesis of prolactin significantly. In addition, the mammary gland tissues of experimental group showed obvious lobulo-alveolar development with milk secretion. Administration of CRE did not cause any signs or symptoms of toxicity which implied that Cyperus rotundus is toxicologically safe. CONCLUSION: This study demonstrates that the aqueous extract of Cyperus rotundus can stimulate milk production in the female rats which may be consequently effective in increasing the lactation of human too.

Foeniculum vulgare Mill: a review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology.

Foeniculum vulgare Mill commonly called fennel has been used in traditional medicine for a wide range of ailments related to digestive, endocrine, reproductive, and respiratory systems. Additionally, it is also used as a galactagogue agent for lactating mothers. The review aims to gather the fragmented information available in the literature regarding morphology, ethnomedicinal applications, phytochemistry, pharmacology, and toxicology of Foeniculum vulgare. It also compiles available scientific evidence for the ethnobotanical claims and to identify gaps required to be filled by future research. Findings based on their traditional uses and scientific evaluation indicates that Foeniculum vulgare remains to be the most widely used herbal plant. It has been used for more than forty types of disorders. Phytochemical studies have shown the presence of numerous valuable compounds, such as volatile compounds, flavonoids, phenolic compounds, fatty acids, and amino acids. Compiled data indicate their efficacy in several in vitro and in vivo pharmacological properties such as antimicrobial, antiviral, anti-inflammatory, antimutagenic, antinociceptive, antipyretic, antispasmodic, antithrombotic, apoptotic, cardiovascular, chemomodulatory, antitumor, hepatoprotective, hypoglycemic, hypolipidemic, and memory enhancing property. Foeniculum vulgare has emerged as a good source of traditional medicine and it provides a noteworthy basis in pharmaceutical biology for the development/formulation of new drugs and future clinical uses.

Nivulian-II a new milk clotting cysteine protease of Euphorbia nivulia latex.

Nivulian-II, new milk clotting cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. Nivulian-II is a monomeric protein with an apparent molecular mass 43670.846 Da. It presents its optimum activity at pH 6.3 and temperature of 50°C. The enzyme was strongly inhibited by common thiol-blocking reagents thereby indicating that it belongs to cysteine protease family. Nivulian-II is a type of glycoprotein and its pI is 3.4. The N-terminal amino acid sequence of Nivulian-II is DFPPNTCCCICC. This sequence showed relatively low homology with several other proteases of Euphorbian plants, suggesting that the isolated enzyme is a new cysteine protease.

Traditional uses, phytochemistry and pharmacology of Ficus carica: a review.

CONTEXT: Ficus carica Linn (Moraceae) has been used in traditional medicine for a wide range of ailments related to digestive, endocrine, reproductive, and respiratory systems. Additionally, it is also used in gastrointestinal tract and urinary tract infection. OBJECTIVE: This review gathers the fragmented information available in the literature regarding morphology, ethnomedicinal applications, phytochemistry, pharmacology, and toxicology of Ficus carica. It also explores the therapeutic potential of Ficus carica in the field of ethnophytopharmacology. MATERIALS AND METHODS: All the available information on Ficus carica was compiled from electronic databases such as Academic Journals, Ethnobotany, Google Scholar, PubMed, Science Direct, Web of Science, and library search. RESULTS: Worldwide ethnomedical uses of Ficus carica have been recorded which have been used traditionally for more than 40 types of disorders. Phytochemical research has led to the isolation of primary as well as secondary metabolites, plant pigment, and enzymes (protease, oxidase, and amylase). Fresh plant materials, crude extracts, and isolated components of Ficus carica have shown a wide spectrum of biological (pharmacological) activities. CONCLUSION: Ficus carica has emerged as a good source of traditional medicine for the treatment of various ailments such as anemia, cancer, diabetes, leprosy, liver diseases, paralysis, skin diseases, and ulcers. It is a promising candidate in pharmaceutical biology for the development/formulation of new drugs and future clinical uses.

Evaluation of hemostatic activity of latex from three Euphorbiaceae species.

ETHNOPHARMACOLOGICAL RELEVANCE: Latices from several plant species of Euphorbiaceae family have been traditionally applied over fresh cuts to stop bleeding and subsequently applied over wounds to enhance healing process. The latex arrested bleeding from fresh wounds by reducing bleeding and whole blood coagulation time which are important indices of hemostatic activity. It has been accepted that hemostatic activity is due to the proteolytic fraction of plant latices. Thus, the present study aimed to assess the clot inducing properties of three Euphorbiaceae plants viz., Euphorbia nivulia Buch.-Ham., Pedilanthus tithymaloides (L.) Poit and Synadenium grantii Hook F. MATERIALS AND METHODS: In the present study, various proteolytic activities namely protease, gelatinase, milk clotting and whole blood clotting assay of the enzyme fraction of latices of Euphorbia nivulia, Pedilanthus tithymaloides and Synadenium grantii have been investigated. The inhibition profile of protease specific inhibitors was assessed. Also, the effects of protein fractions were studied using bleeding/clotting time test of fresh experimentally-induced wounds in mice. RESULTS: Euphorbia nivulia latex protease has noticeable blood clotting activity followed by Pedilanthus tithymaloides and Synadenium grantii. Stem latex protease of Pedilanthus tithymaloides exhibits superior procoagulant activity in different mammal's blood samples viz., Capra hircus, Bubalus bubalis, Ovibos moschatus and Bos indicus. Blood sample of ox was the most sensitive to latex protease than other mammal's blood. Concomitantly, the plant latex protease could significantly reduce whole blood clotting time of human and mice blood samples. CONCLUSION: The protease fraction of latices of Euphorbia nivulia, Pedilanthus tithymaloides and Synadenium grantii possesses phytoconstituents capable of arresting wound bleeding, and accelerating whole blood coagulation process. It suggests good potentiality for use of latex proteases in wound management. Also, the finding of this study showed that the protease enzyme of Pedilanthus tithymaloides has the most potent hemostatic agent.

Identification and characterization of Euphorbia nivulia latex proteins.

The protein profile of latex of Euphorbia nivulia Buch.-Ham. is established. Three new proteins viz., Nivulian-I, II and III have been purified to homogeneity from the latex. The relative molecular masses of Nivulian-I, II and III are 31,486.985, 43,670.846 and 52,803.470 Da respectively. Nivulian-I is a simple type of protein while Nivulian-II and III are glycoproteins. Peptide mass fingerprint analysis revealed peptides of these proteins match with Tubulin alpha-1 chain of Eleusine indica, Maturase K of Banksia quercifolia and hypothetical protein of Zea mays respectively. Tryptic digestion profile of Nivulian-I, II and III, infer the exclusive nature of latex origin proteins and may be new and are additive molecules in the dictionaries of phytoproteins or botany. This is the first of its kind, regarding characterization and validation of Nivulian-I, II and III with respect to peptide sequencing.

Characterization of thermo- and detergent stable antigenic glycosylated cysteine protease of Euphorbia nivulia Buch.-Ham. and evaluation of its ecofriendly applications.

An antigenic glycosylated cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. It exhibits remarkable protease activity in the presence of metal ions, oxidizing agents, organic solvents, and detergents. This enzyme showed potential role in leather processing industry due to its dehairing activity for animal hide without hydrolyzing fibrous proteins, producing, by this way, a better quality product. The enzyme can also be used for silver recovering from X-ray plates. In addition, the stability (temperature and surfactants) and hydrolysis of blood stain data also revealed its application in detergent industries. Agriculturally, this protease finds application in biocontrol process against the infectious management of root knot nematode, Meloidogyne incognita. Biologically, it shows noticeable wound healing, haemostatic and antibacterial activity.

Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.

A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing.