Influence of the expression vector and its elements on recombinant human prolactin synthesis in Escherichia coli; co-directional orientation of replication and transcription is highly critical

2021 ◽  
pp. 106340
Author(s):  
Regina Affonso ◽  
Miriam Fussae Suzuki ◽  
Geraldo Santana Magalhães ◽  
Paolo Bartolini
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Directional Orientation ◽  
Human Prolactin ◽  
Prolactin Synthesis

scholarly journals Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

2021 ◽  
Author(s):  
Regina Affonso ◽  
Miriam Fussae Suzuki ◽  
Geraldo Santana Magalhães ◽  
Paolo Bartolini
Keyword(s):  
Expression Vector ◽  
Heterologous Protein ◽  
Expression System ◽  
Antibiotic Resistance Gene ◽  
Positive Influence ◽  
Individual Study ◽  
E Coli ◽  
Directional Orientation ◽  
Human Prolactin ◽  
The Individual

Abstract The aim of the present work was to define a bacterial expression system that is particularly efficient for the synthesis of human prolactin (hPRL). In previous work, the synthesis of rec-hPRL by the p1813-hPRL vector in E. coli HB2151 was >500 mg/L, while it was much lower now (2.5-4-fold), in the strains RB791 and RRI. The highest positive influence on rec-hPRL synthesis was due to the transcription-replication co-orientation of hPRL cDNA and the ori/antibiotic resistance gene, responsible for up to a ~5-6-fold higher expression yield. In conclusion, this work confirmed that each bacterial strain of E. coli has a genetic set that can allow a different level of heterologous protein synthesis. The individual study of each element indicated that its action critically depends on the reading orientation in which it is located inside the vector: co-directional orientation of replication and transcription, in fact, greatly increased the level of rec-hPRL expression.

Direct Construction and Screening of the Expression Vector of Anaerobic Clostridium in Escherichia coli

2013 ◽  
Vol 19 (5) ◽  
pp. 822-826
Author(s):  
Chunyu LIU ◽  
Yan CHEN ◽  
Jinqun HUANG ◽  
Jianxin PEI ◽  
Hao PANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Direct Construction

scholarly journals Cloning of Salmonella enterica Serovar Enteritidis Fimbrial Protein SefA as a Surface Protein in Escherichia coli Confers the Ability To Attach to Eukaryotic Cell Lines

2009 ◽  
Vol 75 (20) ◽  
pp. 6622-6625 ◽  
Author(s):  
Douglas L. Rank ◽  
Mahdi A. Saeed ◽  
Peter M. Muriana
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Expression Vector ◽  
Surface Protein ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Western Blot Assay ◽  
E Coli ◽  
Fimbrial Protein

ABSTRACT The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

Expression and characterization of the 42 kDa chitinase of the biocontrol fungusMetarhizium anisopliaeinEscherichia coli

2003 ◽  
Vol 49 (11) ◽  
pp. 723-726 ◽  
Author(s):  
César Milton Baratto ◽  
Marcia Vanusa da Silva ◽  
Lucélia Santi ◽  
Luciane Passaglia ◽  
Irene Silveira Schrank ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Metarhizium Anisopliae ◽  
Entomopathogenic Fungus ◽  
Expression Vector ◽  
Hydrolytic Enzymes ◽  
Active Protein ◽  
Chitinase Genes

Albeit Metarhizium anisopliae is the best-characterized entomopathogenic fungus, the role of some hydrolytic enzymes during host cuticle penetration has not yet been established. Three chitinase genes (chit1, chi2, chi3) from Metarhizium have already been isolated. To characterize the chitinase coded by the chit1 gene, we expressed the active protein (CHIT42) in Escherichia coli using a T7-based promoter expression vector. The recombinant protein, CHIT42, is active against glycol chitin and synthetic N-acetylglucosamine (GlcNAc) dimer and tetramer substrates. These activities suggest that the recombinant CHIT42 acts as an endochitinase.Key words: Metarhizium anisopliae, chitinases, chit genes, recombinant protein, enthomopathogenic fungi.

scholarly journals Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

1991 ◽  
Vol 88 (5) ◽  
pp. 1731-1735 ◽  
Author(s):  
S. J. Elledge ◽  
J. T. Mulligan ◽  
S. W. Ramer ◽  
M. Spottswood ◽  
R. W. Davis
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Cdna Expression

Fusion Expression on the Esat-6 and cfp-10 Genes of Mycobacterium bovis in Escherichia coli

2013 ◽  
Vol 421 ◽  
pp. 354-358
Author(s):  
Jia Ming Lin ◽  
Chun Fang Wang ◽  
Jia Ning Guan ◽  
Hong Xia Ma ◽  
Shuang Hou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

Based on the (Gly4Ser)3 linker, theesat-6andcfp-10gene were fused for raising the antigenicity of single antigen. The DNA fragments ofesat-6andcfp-10were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR),and the fusion gene esat-6-cfp-10 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-esat-6-cfp-10. pMD-esat-6-cfp-10 and pET28a (+) were digested byBamHI andEcoRI double enzymes. The purified mpb esat-6-cfp-10 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-esat-6-cfp-10 was constructed. Plasmid containing pET-esat-6-cfp-10 was transformed into competenceEscherichia coliBL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 25 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.

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