scholarly journals Gene expression in Acetabularia. I. Calibration of wheat germ cell-free translation system proteins as internal references for two-dimensional electrophoresis

1982 ◽  
Vol 58 (1) ◽  
pp. 23-33
Author(s):  
R.L. Shoeman ◽  
H.G. Schweiger
Keyword(s):  
Germ Cell ◽  
Isoelectric Point ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Translation ◽  
Translation System ◽  
Two Dimensional ◽  
Free Translation

Modification of existing two-dimensional techniques enables isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis of complex mixtures of proteins to be completed within 8 h. The method was optimized to separate the protein components of a wheat germ cell-free translation system, providing a statistically proven resolution better than 0. 03 of a pH unit for the isoelectric point and 1000 for Mr. Fourteen of the more than 300 proteins separated were characterized with respect to Mr and isoelectric point relative to standard proteins under the same conditions. Stained wheat germ proteins thus serve as internal standards for analysis of in vitro translation products.

Wheat Germ Cell-Free Translation System as a Tool for In vitro Selection of Functional Proteins

2002 ◽  
Vol 5 (6) ◽  
pp. 473-480
Author(s):  
Bentham Science Publisher A.N. Alexandrov ◽  
Bentham Science Publisher V.Yu. Alakhov ◽  
Bentham Science Publisher A.I. Miroshnikov
Keyword(s):  
Germ Cell ◽  
Wheat Germ ◽  
In Vitro Selection ◽  
Translation System ◽  
Free Translation ◽  
Selection Of

scholarly journals Wheat germ in vitro translation to produce one of the most toxic sodium channel specific toxins

2014 ◽  
Vol 34 (4) ◽  
Author(s):  
Wael Gad ◽  
Rahma Ben-Abderrazek ◽  
Khadija Wahni ◽  
Didier Vertommen ◽  
Serge Muyldermans ◽  
...  
Keyword(s):  
Wheat Germ ◽  
Antibody Fragment ◽  
Scorpion Venom ◽  
Biologically Active ◽  
In Vitro Translation ◽  
Translation System ◽  
Single Domain Antibody ◽  
Venom Protein ◽  
Free Translation

A wheat germ embryo derived cell-free translation system expresses a biologically active, highly toxic scorpion venom protein that is fully neutralized by a camel single domain antibody fragment raised against the native scorpion toxin.

scholarly journals Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

2005 ◽  
Vol 4 (11) ◽  
pp. 1951-1958 ◽  
Author(s):  
Felix D. Bastida-Corcuera ◽  
Cheryl Y. Okumura ◽  
Angie Colocoussi ◽  
Patricia J. Johnson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Chemical Mutagenesis ◽  
Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

scholarly journals Proteolytic Processing of Sapovirus ORF1 Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7283-7290 ◽  
Author(s):  
Tomoichiro Oka ◽  
Kazuhiko Katayama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
Tsutomu Kageyama ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Translation System ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Capsid Protein Vp1

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

scholarly journals The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

1986 ◽  
Vol 6 (12) ◽  
pp. 4478-4485 ◽  
Author(s):  
A T Garber ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Protein ◽  
Polyacrylamide Gel ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Alpha Cells ◽  
Two Dimensional ◽  
Isoelectric Ph

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

scholarly journals A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Szilvia Krisztina Nagy ◽  
Brigitta Margit Kállai ◽  
Judit András ◽  
Tamás Mészáros
Keyword(s):  
Protein Expression ◽  
Germ Cell ◽  
Cleavage Site ◽  
Wheat Germ ◽  
In Vitro Translation ◽  
Affinity Tags ◽  
Free Protein ◽  
Target Proteins ◽  
Interaction Studies

Abstract Background Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. Results Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. Conclusions The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

Cell-free synthesis of rat liver zinc-thioneins

1979 ◽  
Vol 57 (7) ◽  
pp. 1030-1035 ◽  
Author(s):  
Choy L. Hew ◽  
Peter E. Penner
Keyword(s):  
Germ Cell ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Zinc Sulfate ◽  
Trichloroacetic Acid ◽  
Wheat Germ ◽  
Translation System ◽  
Glycine Buffer ◽  
Free Translation

The induction of rat liver zinc-thioneins mRNA was studied in a wheat germ cell-free translation system. Liver poly A rich polysomal RNA was isolated from rats which had been injected with zinc sulfate 5 h previously. These RNA preparations stimulated the incorporation of [35S]cystine into trichloroacetic acid insoluble proteins when assayed in the cell-free synthetic system. The translation products were characterized by Sephadex G-75 chromatography in 8 M urea – 50 mM β-mercaptoethanol, by disc gel electrophoresis in 4 M urea – Tris–glycine buffer (pH 9.2), and by peptide fingerprinting with pepsin. These results were identical with authentic rat liver zinc-thioneins. The zinc-thioneins mRNA activity in the control rats, however, was minimal. The stimulation in zinc-thioneins synthesis observed in the cell-free synthesis was similar to the increased synthesis of these polypeptides in vivo.

Production of yeast tRNA (m7G46) methyltransferase (Trm8–Trm82 complex) in a wheat germ cell-free translation system

2008 ◽  
Vol 133 (4) ◽  
pp. 453-460 ◽  
Author(s):  
Keisuke Matsumoto ◽  
Chie Tomikawa ◽  
Takashi Toyooka ◽  
Anna Ochi ◽  
Yoshitaka Takano ◽  
...  
Keyword(s):  
Germ Cell ◽  
Wheat Germ ◽  
Translation System ◽  
Yeast Trna ◽  
Free Translation

scholarly journals Characterization of the Vaccinia Virus A35R Protein and Its Role in Virulence

2006 ◽  
Vol 80 (1) ◽  
pp. 306-313 ◽  
Author(s):  
Rachel L. Roper
Keyword(s):  
Vaccinia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Growth ◽  
Protein Band ◽  
Mutant Virus ◽  
Translation System ◽  
Wild Type ◽  
Infected Cells

ABSTRACT The vaccinia virus A35R gene is highly conserved among poxviruses and encodes a previously uncharacterized hydrophobic acidic protein. Western blotting with anti-A35R peptide antibodies indicated that the protein is expressed early in infection and resolved as a single sharp band of ∼23 kDa, slightly higher than the 20 kDa predicted from its sequence. The protein band appeared to be the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whether expressed in an in vitro transcription/translation system without microsomes or expressed in infected cells, suggesting that it was not glycosylated. A mutant virus with the A35R gene deleted (vA35Δ) formed wild-type-sized plaques on all cell lines tested (human, monkey, mouse, and rabbit); thus, A35R is not required for replication and does not appear to be a host range gene. Although the A35R protein is hydrophobic, it is unlikely to be an integral membrane protein, as it partitioned to the aqueous phase during TX-114 partitioning. The protein could not be detected in virus-infected cell supernatants. A35R localized intracellularly to the virus factories, where the first stages of morphogenesis occur. The vA35Δ mutant formed near-normal levels of the various morphogenic stages of infectious virus particles and supported normal acid-induced fusion of virus-infected cells. Despite normal growth and morphogenesis in vitro, the vA35Δ mutant virus was attenuated in intranasal challenge of mice compared to wild-type and A35R rescue virus. Thus, the intracellular A35R protein plays a role in virulence. The A35R has little homology to any protein outside of poxviruses, suggesting a novel virulence mechanism.

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