Expression of recombinant 35 kDa fragment of VP2 protein of canine parvovirus using Escherichia coli expression system

Background and Aim: Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies. Materials and Methods: The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPOTM expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Results: The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies. Conclusion: These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

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Enhanced production of Canine parvovirus VP2 protein using improved Baculovirus expression system and its immunogenicity

10.21203/rs.3.rs-20865/v1 ◽

2020 ◽

Author(s):

Dao Chang ◽

Yangkun Liu ◽

Yangyang Chen ◽

Xiaomin Hu ◽

Andrey Burov ◽

...

Keyword(s):

Expression System ◽

Baculovirus Expression System ◽

Canine Parvovirus ◽

Full Length ◽

Recombinant Viruses ◽

Vp2 Protein ◽

Enhanced Production ◽

Baculovirus Expression ◽

Yield Production ◽

High Level

Abstract Background : Canine parvovirus (CPV) is now recognized as a serious threat to dog industry worldwide. Vaccination remains the principal tool to control CPV infection. However, due to low yield, production of VP2 protein of CPV in baculovirus expression system remains challenging. The aim of this study was to increase the VP2 protein production by using a improved baculovirus expression system (Multibac) and evaluate the immunogenicity of the purified VP2 protein in mice. Results: The results showed that CPV VP2 protein was successfully expressed in the improved baculovirus expression system efficiently. A high level of expression of the full length VP2 protein was achieved using our modified system. The recombinant virus carrying two copies of VP2 showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein could react with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody with good reactogenicity. The mice were then immunized with purified full length VP2 protein to evaluate its immunogenicity. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. Conclusions: Full length CPV VP2 protein was successfully expressed at high level and purified efficiently. And it stimulated mice to produce high level of antibody. The full length VP2 expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.

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Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation

International Journal of Molecular Sciences ◽

10.3390/ijms22052591 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2591

Author(s):

Pengfei Ma ◽

Jie Li ◽

Lei Qi ◽

Xiuzhu Dong

Keyword(s):

Heat Shock ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Small Heat Shock Protein ◽

Phase Cell ◽

Molecular Weights ◽

Oxidative Inactivation ◽

Methionine Residues ◽

Shock Proteins ◽

And Function

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70–90% and 86–100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an “oxidant sink” effect, i.e., to consume the oxidants in environments via aspartate oxidation

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Effect of detergents and chemicals on purified vaccinia virus: analysis by SDS polyacrylamide gel electrophoresis and electron microscopy

Canadian Journal of Microbiology ◽

10.1139/m77-036 ◽

1977 ◽

Vol 23 (3) ◽

pp. 240-252 ◽

Cited By ~ 4

Author(s):

J. Boisvert ◽

T. Yamamoto

Keyword(s):

Electron Microscopy ◽

Vaccinia Virus ◽

Gel Electrophoresis ◽

Alkali Treatment ◽

Guanidine Hydrochloride ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Ammonium Bromide ◽

Molecular Weights ◽

Viral Particles

Vaccinia virus particles were dissociated into their constituent polypeptides and analysed by sodium dodecyl sulfate (SDS) gel electrophoresis. Thirty-three distinct polypeptide bands were identified and their molecular weights ranged between 11 000 and 150 000 daltons.Specific staining of gels containing polypeptides of dissociated virions revealed the presence of eight glycopeptides. No lipopeptides were detected.Analysis of chemical extracts (urea, guanidine hydrochloride, and alkali treatment) of the virus by SDS gel electrophoresis indicated that a total of 10 to 14 different polypeptides ranging in molecular weights from 11 000 to 70 000 daltons were solubilized.Analysis of detergent extracts and of the remains of extracted viral particles has shown that the detergent Nonidet P-40 (NP-40) solubilized a total of 11 polypeptides of which 6 were glycopeptides. The other detergents sodium deoxycholate (SDC) and cetyl trimethyl ammonium bromide (CTAB) were not as selective, both solubilizing more than 25 of the polypeptides composing the virus. Gel electrophoresis results also indicated that most of the small molecular weight (11 000–70 000 daltons) polypeptides were readily solubilized by NP-40, SDC, and CTAB, while those with molecular weights of 70 000 daltons and higher were not well solubilized.The effects of detergents were also analysed by electron microscopy. Evidence was obtained for subpopulations of viral particles having different susceptibility to detergent extraction.

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Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5177-5186.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5177-5186

Author(s):

J Zhang ◽

S T Jacob

Keyword(s):

Ribosomal Dna ◽

Core Promoter ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Affinity Column ◽

Mobility Shift ◽

Enhancer Element ◽

Molecular Weights ◽

The Core ◽

Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

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Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

Parasitology Open ◽

10.1017/pao.2017.14 ◽

2017 ◽

Vol 3 ◽

Cited By ~ 6

Author(s):

A. R. Escalona-Montaño ◽

R. Pérez-Montfort ◽

N. Cabrera ◽

R. Mondragón-Flores ◽

D. E. Vélez-Ramírez ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Protein Phosphatase ◽

Protein Concentration ◽

Leishmania Major ◽

Polyclonal Antibodies ◽

Divalent Cations ◽

Tyrosine Phosphatase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Phosphatase Inhibitors

AbstractThe main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes ofLeishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinantLmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinantLmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in differentLeishmaniaspecies; theLmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

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Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.252-256.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 252-256 ◽

Cited By ~ 19

Author(s):

Katsuichi Saito ◽

Kazuya Kondo ◽

Ichiro Kojima ◽

Atsushi Yokota ◽

Fusao Tomita

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Extracellular Enzyme ◽

Molecular Weights ◽

Sds Page ◽

Monomeric Structure ◽

Ph Range ◽

Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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Characterization of proteins from the cytoskeleton of Giardia lamblia

Journal of Cell Science ◽

10.1242/jcs.59.1.81 ◽

1983 ◽

Vol 59 (1) ◽

pp. 81-103 ◽

Cited By ~ 1

Author(s):

R. Crossley ◽

D.V. Holberton

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulphate ◽

Giardia Lamblia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Molecular Size ◽

Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

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Subunit studies of coupling factor 1 of bean chloroplasts

Canadian Journal of Biochemistry ◽

10.1139/o76-069 ◽

1976 ◽

Vol 54 (5) ◽

pp. 481-487 ◽

Cited By ~ 1

Author(s):

M. P. Silvanovich ◽

R. D. Hill

Keyword(s):

Amino Acid ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Coupling Factor ◽

Leaf Extracts ◽

Molecular Weights ◽

Major Species ◽

Chloroplast Coupling Factor ◽

Coupling Factors

A bean chloroplast coupling factor (CF1) with latent Ca2+-dependent ATPase activity was studied. Immunodiffusion of bean (Phaseolus vulgaris) chloroplast and etioplast coupling factors and spinach coupling factor against antiserum to spinach coupling factor showed partial identity of the bean coupling factor with that of spinach. An immunoelectrophoretic comparison, under dissociating conditions, of bean leaf extracts and spinach extracts containing CF1 subunits (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) gave identical results for both extracts. At least six distinct polypeptide species were found. The major species had molecular weights of 42 000, 59 000 and 63 000 daltons. Amino acid analysis of electrophoretically purified bean CF1 gave results similar to those published for spinach CF1.

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Effects of Wheat Bug (Eurygasterspp. andAeliaspp.) Infestation in Preharvest Period on Wheat Technological Quality and Gluten Composition

The Scientific World JOURNAL ◽

10.1155/2014/148025 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Aleksandra M. Torbica ◽

Jasna S. Mastilović ◽

Milica M. Pojić ◽

Žarko S. Kevrešan

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Wheat Gluten ◽

Sodium Dodecyl ◽

Deformation Energy ◽

Wheat Varieties ◽

Gluten Proteins ◽

Molecular Weights ◽

Sds Page ◽

Technological Quality ◽

Gluten Index

The effects of wheat bug infestation (Eurygasterspp. andAeliaspp.) on the composition of wheat gluten proteins and its influence on flour technological quality were investigated in the present study. Wheat samples of six wheat varieties, collected from two localities in northern Serbia, were characterized by significantly different level of wheat bug infestation. Composition of wheat gluten proteins was determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), while the selected parameters of technological quality were determined according to standard and modified empirical rheological methods (Farinograph, Extensograph, Alveograph, and Gluten Index). The surface morphology of the selected samples was viewed using scanning electron microscopy (SEM). Wheat from wheat bug-infested locality regardless of the variety had deteriorated technological quality expressed with higher Farinograph softening degree, lower or immeasurable Extensograph energy, and Alveograph deformation energy. The most important changes in the gluten proteins composition of bug-infested wheat were related to gliadin subunits with molecular weights below 75 kDa, which consequently caused deterioration of uniaxial and biaxial extensibility and dough softening during mixing.

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