AnEscherichia coli plasmid vector system for production of streptavidin fusion proteins: Expression and bioselective adsorption of streptavidin-?-galactosidase

1994 ◽  
Vol 44 (11) ◽  
pp. 1348-1354 ◽  
Author(s):  
Marie K. Walsh ◽  
Harold E. Swaisgood
Keyword(s):  
Fusion Proteins ◽  
Plasmid Vector ◽  
Vector System ◽  
Coli Plasmid

Molecular cloning of polyoma virus DNA in Escherichia coli: plasmid vector system

Science ◽  
1979 ◽  
Vol 203 (4383) ◽  
pp. 883-887 ◽  
Author(s):  
M. Israel ◽  
H. Chan ◽  
W. Rowe ◽  
M. Martin
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Plasmid Vector ◽  
Polyoma Virus ◽  
Vector System ◽  
Coli Plasmid

scholarly journals Two-Plasmid Vector System for Independently Controlled Expression of Green and Red Fluorescent Fusion Proteins in Staphylococcus aureus

2013 ◽  
Vol 79 (9) ◽  
pp. 3133-3136 ◽  
Author(s):  
Anthony J. Brzoska ◽  
Neville Firth
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Vector System ◽  
Red Fluorescent Protein ◽  
Filament Formation ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTWe have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions inStaphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

Comparison of intron-dependent and intron-independent gene expression

1988 ◽  
Vol 8 (10) ◽  
pp. 4395-4405
Author(s):  
A R Buchman ◽  
P Berg
Keyword(s):  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Fold Increase ◽  
Simian Virus ◽  
Transcription Unit ◽  
Vector System ◽  
Beta Globin ◽  
Animal Cells ◽  
Transcriptional Enhancer ◽  
Kinase Gene

Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.

Generation of transducible versions of transcription factors Oct4 and Sox2

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Manal Bosnali ◽  
Frank Edenhofer
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Genetic Modification ◽  
Fusion Proteins ◽  
Embryonic Stem ◽  
Vector System ◽  
Target Cells ◽  
Protein Transduction ◽  
Cellular Delivery

Abstract The transcription factors Oct4 and Sox2 are two of the main regulators of pluripotency in embryonic stem cells. Since the importance of non-genetic modification is continually increasing, particularly for therapeutic application of manipulated cells, the aim of the present study was to generate cell-permeant Oct4 and Sox2 proteins for the direct cellular delivery of active proteins. Protein transduction allowing cellular manipulation to circumvent genetic modification of target cells has recently been developed. We present a new expression vector system, pSESAME, that facilitates the generation of transducible proteins. Using pSESAME, both Oct4 and Sox2 were genetically fused with a TAT protein transduction domain that promotes cellular penetration. The recombinant purified Oct4 and Sox2 fusion proteins display DNA-binding properties comparable to their endogenous counterparts, and exhibit cellular entry and the ability to modulate the transcriptional machinery maintaining pluripotency of mouse embryonic stem cells. In a rescue assay we demonstrate that transducible Oct4 and Sox2 fusion proteins can compensate knockdown of Pou5f1 and Sox2, respectively. This study provides powerful tools for the modulation of stem cell properties without genetic interference.

scholarly journals INTRODUCTION OF β-GLUCURONIDASE GENE INTO GINSENG (PANAX CINSENG) BY A TI-PLASMID VECTOR SYSTEM AND ITS EXPRESSION

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 621e-621
Author(s):  
Jang R. Liu ◽  
Haeng S. Lee ◽  
Suk W. Kim ◽  
Hyo W. Lee
Keyword(s):  
Somatic Embryos ◽  
Binary Vector ◽  
Zygotic Embryo ◽  
Plasmid Vector ◽  
Vector System ◽  
Selection Marker ◽  
35S Promoter ◽  
Dichlorophenoxyacetic Acid ◽  
Gus Gene ◽  
2,4 Dichlorophenoxyacetic Acid

β-Glucuronidase (GUS) gene of Escherichia coli was introduced into ginseng cells by an Agrobacterium binary vector system and expressed in somatic embryos derived from the cells. A binary vector pBI121 carrying CaMV 35S promoter-GUS gene fusion and a neomycin phosphotransferase gene as selection marker was transferred into Agrobacterium tumefaciens LBA4404. Zygotic embryo cotyledonary segments were co-cultivated with A. tumefaciens and transferred to the medium containg 1 mg 2,4-dichlorophenoxyacetic acid/liter, 0.5 mg kinetin/liter, and 100 mg kanamycin/liter. Kanamycin-resistant calli were formed after 3 to 4 weeks of culture. Southern analysis confirmed the resistant calli were transformed with GUS gene. High GUS activities were detected in somatic embryos developed from the calli.

scholarly journals Transformation and Expression of a ClonedfimA Gene in Porphyromonas gingivalis

1999 ◽  
Vol 67 (4) ◽  
pp. 2013-2018 ◽  
Author(s):  
Yusuke Takahashi ◽  
Daisuke Kato ◽  
Nobushiro Hamada ◽  
Hisashi Yoshimoto ◽  
Toshio Umemoto
Keyword(s):  
Electron Microscopy ◽  
Porphyromonas Gingivalis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasmid Vector ◽  
Amino Acid Sequences ◽  
Vector System ◽  
Immunogold Electron Microscopy ◽  
Amino Terminal ◽  
Fimbrial Subunit

ABSTRACT The Porphyromonas gingivalis fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene, fimA 381, encoding the fimbrial subunit of P. gingivalis 381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned fimA 381 gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the fimA 381 gene into the plasmid vector pYH420 and transformed into the restriction-deficient P. gingivalis host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host’s native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the fimA 381 gene. These results suggested that transformation of fimA gene from a different strain ofP. gingivalis followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.

scholarly journals Comparison of intron-dependent and intron-independent gene expression.

1988 ◽  
Vol 8 (10) ◽  
pp. 4395-4405 ◽  
Author(s):  
A R Buchman ◽  
P Berg
Keyword(s):  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Fold Increase ◽  
Simian Virus ◽  
Transcription Unit ◽  
Vector System ◽  
Beta Globin ◽  
Animal Cells ◽  
Transcriptional Enhancer ◽  
Kinase Gene

Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.

Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

1983 ◽  
Vol 3 (1) ◽  
pp. 143-146
Author(s):  
Aleem Siddiqui
Keyword(s):  
Hepatitis B ◽  
Surface Antigen ◽  
Recombinant Plasmid ◽  
Cultured Cells ◽  
Hepatitis B Surface Antigen ◽  
Plasmid Vector ◽  
Vector System ◽  
New Host ◽  
Virus Surface ◽  
Gene Coding

By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

Efficient production of complement (C3d)3 fusion proteins using the baculovirus expression vector system

2005 ◽  
Vol 304 (1-2) ◽  
pp. 158-173 ◽  
Author(s):  
Denise V. Barrault ◽  
Michael Steward ◽  
Vivienne F. Cox ◽  
Richard A.G. Smith ◽  
Andrew M. Knight
Keyword(s):  
Fusion Proteins ◽  
Expression Vector ◽  
Vector System ◽  
Efficient Production ◽  
Baculovirus Expression Vector System ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector
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