scholarly journals Development of a Novel Chloramphenicol Resistance Expression Plasmid Used for Genetic Complementation of a fliG Deletion Mutant in Treponema denticola

2004 ◽  
Vol 72 (9) ◽  
pp. 5493-5497 ◽  
Author(s):  
Linda L. Slivienski-Gebhardt ◽  
Jacques Izard ◽  
William A. Samsonoff ◽  
Ronald J. Limberger
Keyword(s):  
Treponema Pallidum ◽  
Treponema Denticola ◽  
High Level Expression ◽  
Expression Plasmid ◽  
Chloramphenicol Resistance ◽  
Expression Of Genes ◽  
Colony Spreading ◽  
High Level ◽  
Resistance Expression

ABSTRACT A new expression plasmid containing the fla operon promoter and a staphylococcal chloramphenicol resistance gene, was constructed to help assess the role of fliG in Treponema denticola motility. Deletion of fliG resulted in a nonmotile mutant with a markedly decreased number of flagellar filaments. Wild-type fliG genes from T. denticola and from Treponema pallidum were cloned into this expression plasmid. In both cases, the gene restored the ability of the mutant to gyrate its cell ends and enabled colony spreading in agarose. This shuttle plasmid enables high-level expression of genes in T. denticola and possesses an efficient selectable marker that provides a new tool for treponemal genetics.

scholarly journals Structural and functional analysis of a promoter of the human granulin/epithelin gene

1996 ◽  
Vol 319 (2) ◽  
pp. 441-447 ◽  
Author(s):  
Vijay BHANDARI ◽  
Rachael DANIEL ◽  
Pheng Siew LIM ◽  
Andrew BATEMAN
Keyword(s):  
Promoter Activity ◽  
Biologically Active ◽  
Gene Products ◽  
High Level Expression ◽  
Haematopoietic Cells ◽  
Molecular Masses ◽  
Chloramphenicol Acetyltransferase Gene ◽  
High Level

Granulins (grns) or epithelins (epis) are peptides with molecular masses of approx. 6 kDa that modulate the growth of cells. The precursor for the grns/epis, which might itself be biologically active, is a secreted glycoprotein containing multiple repeats of the grn/epi motif. Grn/epi mRNA occurs widely in vivo, particularly in tissues rich in epithelial and haematopoietic cells. To understand better the role of the gene products for grn/epi it is important to determine the patterns of grn/epi gene expression and how this is regulated. To assist in this we have obtained the 5´ sequence of the human grn/epi gene, and using chimaeras of the grn/epi -5´ sequence and the chloramphenicol acetyltransferase gene we have shown a strong promoter activity associated with the 5´ sequence of the human grn/epi gene. We have further delineated regions of the 5´ sequence that confer high-level expression on the chimaeric gene.

scholarly journals Role of AmpR in the High Expression of the Plasmid-Encoded AmpC β-Lactamase CFE-1

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Ryuichi Nakano ◽  
Akiyo Nakano ◽  
Hisakazu Yano ◽  
Ryoichi Okamoto
Keyword(s):  
High Resistance ◽  
High Expression ◽  
Regulator Gene ◽  
High Level Expression ◽  
Expression Levels ◽  
High Level ◽  
Unique Plasmid

ABSTRACT CFE-1 is a unique plasmid-encoded AmpC β-lactamase with the regulator gene ampR. It imparts high resistance to most cephalosporins with constitutive high-level β-lactamase activity. CFE-1 is a unique plasmid-encoded AmpC β-lactamase with the regulator gene ampR. It imparts high resistance to most cephalosporins with constitutive high-level β-lactamase activity. Here, the β-lactamase activities and expression levels of ampC with or without ampR were investigated. Results suggested that the resistance of CFE-1 to cephalosporins is caused by a substitution in AmpR, in which the Asp at position 135 is modified to Ala to allow the constitutive high-level expression (derepression) of ampC.

scholarly journals Neuropilin-2 Is Associated With Increased Hepatoblastoma Cell Viability and Motility

2021 ◽  
Vol 9 ◽  
Author(s):  
Katja Eloranta ◽  
Ruth Nousiainen ◽  
Stefano Cairo ◽  
Mikko P. Pakarinen ◽  
David B. Wilson ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Migration And Invasion ◽  
High Level Expression ◽  
Liver Malignancy ◽  
Rna Analysis ◽  
Cytoskeleton Remodeling ◽  
High Level ◽  
Tumor Types

The neuropilins NRP1 and NRP2 are multifunctional glycoproteins that have been implicated in several cancer-related processes including cell survival, migration, and invasion in various tumor types. Here, we examine the role of neuropilins in hepatoblastoma (HB), the most common pediatric liver malignancy. Using a combination of immunohistochemistry, RNA analysis and western blotting, we observed high level expression of NRP1 and NRP2 in 19 of 20 HB specimens and in a majority of human HB cell lines (HUH6 and five cell lines established from patient-derived xenografts) studied but not in normal hepatocytes. Silencing of NRP2 expression in HUH6 and HB-282 HB cells resulted in decreased cell viability, impaired cytoskeleton remodeling, and reduced cell motility, suggesting that NRP2 contributes to the malignant phenotype. We propose that neuropilins warrant further investigation as biomarkers of HB and potential therapeutic targets.

scholarly journals A Dominant Interfering Bub1 Mutant Is Insufficient To Induce or Alter Thymic Tumorigenesis In Vivo, Even in a Sensitized Genetic Background

2005 ◽  
Vol 25 (17) ◽  
pp. 7796-7802 ◽  
Author(s):  
Dale O. Cowley ◽  
Ginger W. Muse ◽  
Terry Van Dyke
Keyword(s):  
Mitotic Spindle ◽  
Genetic Background ◽  
Cultured Cells ◽  
Spindle Checkpoint ◽  
Transgenic Animals ◽  
Dominant Negative ◽  
High Level Expression ◽  
High Level

ABSTRACT Aneuploidy is a common feature of human tumors, often correlating with poor prognosis. The mitotic spindle checkpoint is thought to play a major role in aneuploidy suppression. To investigate the role of the spindle checkpoint in tumor suppression in vivo, we developed transgenic mice in which thymocytes express a dominant interfering fragment of Bub1, a kinase regulator of the spindle checkpoint. We report that, despite high-level expression of dominant-negative Bub1 (Bub1DN), a protein known to inhibit spindle checkpoint activity in cultured cells, thymocytes show no evidence of spindle checkpoint impairment. Transgenic animals also failed to show an increased predisposition to spontaneous tumors. Moreover, the Bub1DN transgene failed to alter the timing or characteristics of thymic lymphoma development in p53 heterozygous or homozygous null backgrounds, indicating that the lack of tumorigenesis is not due to suppression by p53-dependent checkpoints. These results indicate that overexpression of a Bub1 N-terminal fragment is insufficient to impair the spindle checkpoint in vivo or to drive tumorigenesis in the highly susceptible murine thymocyte system, either alone or in combination with G1 checkpoint disruption.

scholarly journals High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

Membranes ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 715
Author(s):  
Agnieszka Chytła ◽  
Weronika Gajdzik-Nowak ◽  
Agnieszka Biernatowska ◽  
Aleksander F. Sikorski ◽  
Aleksander Czogalla
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
High Yield ◽  
High Level Expression ◽  
Lateral Membrane ◽  
Protein Membrane ◽  
High Level ◽  
Lateral Organization

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

scholarly journals Effect of ecd1 mutation on the expression of genes mapped at the Drosophila melanogaster 3C11-12 intermoult puff

1992 ◽  
Vol 59 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Maria Furia ◽  
Pier Paolo D'avino ◽  
Filomena A. Digilio ◽  
Stefania Crispi ◽  
Ennio Giordano ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Steroid Hormone ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Expression Of Genes ◽  
High Level

SummaryThe Drosophila melanogasterecd1 mutation causes a severe temperature-sensitive deficiency in the titre of the steroid hormone eedysone. This mutation was used to investigate the role of eedysone in both the transcription of the genes mapped at the 3C11–12 intermoult puff region and the puff formation. Thoroughly synchronized ecd1 larvae were shifted to the non-permissive temperature at various times of the development; after 24 or 48 h, the levels of the transcripts derived from Sgs-4, Pig-1 and ng-1, the three genes located at the 3C11–12 polytene bands, were determined. The results showed that the levels of the transcripts encoded by Pig-1 and ng-1 are unaffected by the drop in the ecdysone titre occurring in non-permissive conditions whereas the amount of Sgs-4 mRNA is greatly reduced. These data clearly indicate that transcription of the three genes mapped within the puff region is affected differently by the hormone. Furthermore, ecd1 larvae cultured at the non-permissive temperature show a prominent puff at the 3C11–12 polytene bands, indicating that eedysone is not essential for puff induction and that puff size is not simply correlated with high-level Sgs-4 transcription.

scholarly journals High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection": The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Biochemistry ◽  
1994 ◽  
Vol 33 (40) ◽  
pp. 12329-12339 ◽  
Author(s):  
Sasa Frank ◽  
Krisztina Krasznai ◽  
Srdan Durovic ◽  
Eva-Maria Lobentanz ◽  
Hans Dieplinger ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
High Level Expression ◽  
Apolipoprotein A ◽  
High Level

Expression, Purifi cation, and Characterization of Functional Recombinant Human Daintain/AIF-1 in Escherichia coli

2010 ◽  
Vol 65 (11-12) ◽  
pp. 726-732 ◽  
Author(s):  
Wei Wang ◽  
Bin Huang ◽  
Zhi-Guo Feng ◽  
Xiao-Ping Chen ◽  
Win-Xin Tang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Purification And Characterization ◽  
High Level Expression ◽  
Expression Plasmid ◽  
Recombinant Peptide ◽  
Increase Blood Glucose ◽  
Sds Page ◽  
High Level ◽  
Mcf 7

Daintain/AIF-1 was identified from injured rat carotid arteries and porcine intestine in the mid 1990s. It is involved in autoimmune disorders, chronic rejection of allografts, gliomas, and breast cancer. Since it is convenient and economical to obtain such a peptide biologically, in this study, we describe the expression, purification, and characterization of recombinant human daintain/AIF-1 (rhdaintain/AIF-1). The backbone of vector pET32a, a high-level expression plasmid, was used to construct the pET32a-daintain/AIF-1 plasmid for daintain/AIF-1 expression in Escherichia coli. The recombinant daintain/AIF-1 protein was solubly expressed in the BL21 (DE3) strain and was purifi ed by Ni2+ affinity chromatography. After purification, the recombinant protein showed the expected size of 18 kDa on Tricine-SDS-PAGE gels which was further confirmed by Western blotting. A total of 34.0 mg of high purity (over 98%) rhdaintain/AIF-1 was obtained from 1 L culture. The recombinant peptide was able to increase blood glucose elimination rates and enhance the proliferation of human MCF-7 cells. These results suggest that biological activity of the recombinant peptide was preserved after purification

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