scholarly journals Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System

2008 ◽  
Vol 74 (18) ◽  
pp. 5854-5856 ◽  
Author(s):  
D. M. Villarreal ◽  
C. L. Phillips ◽  
A. M. Kelley ◽  
S. Villarreal ◽  
A. Villaloboz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Human Hemoglobin ◽  
Plesiomonas Shigelloides ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Heme Transport ◽  
Protein Folds ◽  
Hemoglobin Production

ABSTRACT To produce recombinant hemoglobin in Escherichia coli, sufficient intracellular heme must be present, or the protein folds improperly and is degraded. In this study, coexpression of human hemoglobin genes and Plesiomonas shigelloides heme transport genes enhanced recombinant hemoglobin production in E. coli BL21(DE3) grown in medium containing heme.

scholarly journals Development of a Method To Produce Hemoglobin in a Bioreactor Culture of Escherichia coli BL21(DE3) Transformed with a Plasmid Containing Plesiomonas shigelloides Heme Transport Genes and Modified Human Hemoglobin Genes

2011 ◽  
Vol 77 (18) ◽  
pp. 6703-6705 ◽  
Author(s):  
B. J. Z. Smith ◽  
P. Gutierrez ◽  
E. Guerrero ◽  
C. J. Brewer ◽  
D. P. Henderson
Keyword(s):  
Escherichia Coli ◽  
Dry Weight ◽  
Bioreactor Culture ◽  
Human Hemoglobin ◽  
Plesiomonas Shigelloides ◽  
Content Type ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Heme Transport ◽  
A Cell

ABSTRACTWe describe a method for production of recombinant human hemoglobin byEscherichia coligrown in a bioreactor.E. coliBL21(DE3) transformed with a plasmid containing hemoglobin genes andPlesiomonas shigelloidesheme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.

High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia coli

1998 ◽  
Vol 64 (5) ◽  
pp. 1589-1593 ◽  
Author(s):  
Michael J. Weickert ◽  
Izydor Apostol
Keyword(s):  
Escherichia Coli ◽  
Error Rates ◽  
High Fidelity ◽  
Human Hemoglobin ◽  
Heterologous Proteins ◽  
Expression Systems ◽  
High Level Expression ◽  
E Coli ◽  
Translation Error ◽  
High Level

ABSTRACT Coexpression of di-α-globin and β-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ∼20% of the level of E. colisoluble proteins also resulted in equivalent translational fidelity.

scholarly journals Induced immune response of Escherichia coli BL21 expressing recombinant MSP1a and MSP1b proteins of Anaplasma marginale

2009 ◽  
Vol 52 (spe) ◽  
pp. 113-120 ◽  
Author(s):  
Katia Tamekuni ◽  
Marilda Carlos Vidotto ◽  
Samuel Rodrigues Felix ◽  
Michelle Igarashi ◽  
João Luis Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Response ◽  
Western Blot ◽  
Molecular Mass ◽  
Outer Membrane ◽  
Humoral Response ◽  
Anaplasma Marginale ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Molecular Masses

This work aims to evaluate the potential of immunization with E. coli BL21 expressing the recombinant rMSP1a and rMSP1b proteins of Anaplasma marginale. E. coli BL21 was transformed with recombinant plasmids pET102/msp1α and pET101/msp1β, and rMSP1a and rMSP1b were expressed after induction by IPTG. BALB/c mice were vaccinated with formolized BL21/rMSP1a and BL21/rMSP1b, and the production in mice sera of whole IgG was determined by ELISA. The mice immunized with BL21/rMSP1a showed a better humoral response for whole IgG when compared to the mice immunized with BL21/rMSP1b; these mice exhibited a small response after the second vaccination. Sera of mice immunized with BL21/rMSP1a reacted via western blot with BL21 and rMSP1a, with molecular masses varying from 70 to 105 kDa. Sera of mice immunized with BL21/rMSP1b reacted with BL21 and rMSP1b with a molecular mass of 100 kDa. These results demonstrate that BL21 containing rMSP1a and rMSP1b in the outer membrane were able to produce an immune response in mice, reinforcing its use in vaccine models against bovine anaplasmosis.

scholarly journals The Hydantoin Transport Protein from Microbacterium liquefaciens

2006 ◽  
Vol 188 (9) ◽  
pp. 3329-3336 ◽  
Author(s):  
Shun'ichi Suzuki ◽  
Peter J. F. Henderson
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Membrane Fraction ◽  
Transport Protein ◽  
Host Strain ◽  
Expression Plasmid ◽  
Ph Optimum ◽  
Inner Membrane ◽  
E Coli ◽  
Micromolar Range

ABSTRACT The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His6 tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent Km values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the l isomer over the d isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.

scholarly journals 2-Deoxy-d-galactose, a substrate for the galactose-transport system of Escherichia coli

1977 ◽  
Vol 168 (1) ◽  
pp. 15-22 ◽  
Author(s):  
P J F Henderson ◽  
R A Giddens
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Transport Systems ◽  
Mutual Inhibition ◽  
E Coli ◽  
System 2 ◽  
Galactose Transport ◽  
Proton Uptake ◽  
Inhibition Studies ◽  
Lactose Transport

The following observations showed that 2-deoxy-D-galactose is a useful tool for the isolation and elucidation of the activity of one system for galactose uptake into Escherichia coli. 1. 2-Deoxygalactose, which is not a substrate for growth of E. coli, was transported into strains of the organism induced for galactose transport. 2. By using appropriate mutants it was shown that 2-deoxygalactose is a much better substrate for the galactose-transport system than for the methyl galactoside-transport system. This was confirmed by the results of mutual inhibition studies with substrates of each transport system. 3. The glucose-, arabinose- or lactose-transport systems did not effect significant transport of 2-deoxygalactose. 4. Like other substrates of the galactose-transport system, 2-deoxygalactose promoted effective proton uptake into de-energized suspensions of appropriate E. coli strains. 5. The S183 series of E. coli mutants were found to contain a constitutive galactose-transport system, if 2-deoxygalactose transport is used as one criterion for such activity.

scholarly journals Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coliCultures by Use of the Hemolysin System

2000 ◽  
Vol 66 (11) ◽  
pp. 5024-5029 ◽  
Author(s):  
Luis A. Fernández ◽  
Isabel Sola ◽  
Luis Enjuanes ◽  
Víctor de Lorenzo
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Culture Media ◽  
Single Chain ◽  
Extracellular Medium ◽  
Simple Method ◽  
E Coli ◽  
Single Chain Fv ◽  
Bacterial Cytoplasm ◽  
Culture Supernatants

ABSTRACT A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

PRODUKSI REKOMBINAN SEFALOSPORIN ASILASE SEBAGAI BIOKATALIS UNTUK PRODUKSI ASAM 7-AMINOSEFALOSPORANAT

2017 ◽  
Vol 4 (1) ◽  
pp. 28 ◽  
Author(s):  
Bima Wedana Isdiyono ◽  
Dudi Hardianto ◽  
Fransiskus Xaverius Ivan
Keyword(s):  
Escherichia Coli ◽  
Induction Time ◽  
Acid Oxidase ◽  
Enzymatic Method ◽  
Amino Acid Oxidase ◽  
Two Stage ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Cephalosporin Acylase ◽  
The One

Production of Cephalosporin Acylase Recombinant as Biocatalyst for 7-Aminocephalosporanic Acid Production7-aminocephalosporanic acid (7-ACA) is a precursor for the production of semisynthetic cephalosporin derivatives. The enzymatic 7-ACA production can use two-stage and one-step enzymatic methods. Two-stage enzymatic method uses D-amino acid oxidase (DAAO) enzyme to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA) in the first stage and glutaryl-7-aminocephalosporanic acid acylase to produce 7-ACA in the second stage. The one-stage enzymatic method using cephalosporin acylase (CPC acylase) converts the CPC to 7-ACA directly. The aim of this research was to produce recombinant CPC acylase in Escherichia coli BL21(DE3). Transformantion culture E. coli BL21(DE3) was induced with concentrations of IPTG 0; 0.25; 0.5; 0.75; 1; 2 mM for 5 hours. The induction time of IPTG was determined at 0, 1, 2, 3, 4, and 5 hours. The results showed that CPC acylase produced by E. coli BL21(DE3) with optimum condition of CPC acylase production was 0.5 mM IPTG and optimal induction time of IPTG was 5 hours.Keywords: Cephalosporin, cephalosporin acylase, 7-ACA, protein expression, Escherichia coli BL21(DE3) ABSTRAKAsam 7-aminosefalosporanat (7-ACA) merupakan prekursor untuk produksi turunan sefalosporin semisintetik. Produksi 7-ACA secara enzimatik dapat menggunakan metode dua tahap dan satu tahap enzimatik. Metode enzimatik secara dua tahap menggunakan enzim asam D-amino oksidase (DAAO) untuk menghasilkan asam glutaril-7-aminosefalosporinat (GL-7-ACA) pada tahap pertama dan menggunakan asam glutaril-7-aminosefalosporinat asilase untuk menghasilkan 7-ACA pada tahap kedua. Metode enzimatik satu tahap dengan sefalosporin asilase (CPC asilase) mengubah CPC menjadi 7-ACA secara langsung. Tujuan penelitian adalah memproduksi rekombinan CPC asilase di dalam sel Escherichia coli BL21(DE3). Kultur Transforman E. coli BL21(DE3) diinduksi dengan konsentrasi IPTG 0; 0,25; 0,5; 0,75; 1; 2 mM selama 5 jam. Waktu induksi IPTG ditentukan pada 0, 1, 2, 3, 4 dan 5 jam. Hasil penelitian menunjukan bahwa CPC asilase diproduksi oleh E. coli BL21(DE3) dengan kondisi optimal produksi CPC asilase adalah konsentrasi IPTG 0,5 mM dan waktu induksi IPTG optimal adalah 5 jam.

scholarly journals Generation of two New Macrolactones through Sequential Biotransformation of Dihydroresorcylide

2011 ◽  
Vol 6 (2) ◽  
pp. 1934578X1100600
Author(s):  
Jia Zeng ◽  
Jonathan Valiente ◽  
Jixun Zhan
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Natural Product ◽  
Beauveria Bassiana ◽  
Natural Product Biosynthesis ◽  
Whole Cell ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Engineered Escherichia Coli

Biotransformation is an effective method to generate new derivatives from natural products. Combination of various enzymes or whole-cell biocatalysts creates new opportunities for natural product biosynthesis. Dihydroresorcylide (1) is a phytotoxic macrolactone from Acremonium aeae. It was first chlorinated at C-11 by an engineered Escherichia coli BL21-CodonPlus (DE3)-RIL/pJZ54 strain that overexpresses a fungal flavin-dependent halogenase, and subsequently glycosylated at 12-OH by Beauveria bassiana ATCC 7159, giving rise to a novel derivative, 11-chloro-4′- O-methyl-12- O-β-D-glucosyl-dihydroresorcylide (3). Although 1 can be converted into a new 4′- O-methyl-glucosylated derivative 4 by B. bassiana, this product cannot be further chlorinated by E. coli BL21-CodonPlus (DE3)-RIL/pJZ54 to afford 3. The sequence of these two biotransformation steps was thus restricted and not interchangeable. This sequential biotransformation approach can be applied to other structurally similar natural products to create novel derivatives.

scholarly journals EXPRESSION OF GLUTARYL7-AMINOCEPHALOSPORANIC ACID ACYLASE IN ESCHERICHIA COLI BL21(DE3) AND IMMOBILIZATION OF RECOMBINANT ENZYME ON NANOPOROUS MATERIALS

2018 ◽  
Vol 54 (4A) ◽  
pp. 123
Author(s):  
Vu Thi Hanh
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Expression Vector ◽  
Nanoporous Materials ◽  
Acid Oxidase ◽  
Nano Materials ◽  
Amino Acid Oxidase ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Gla Gene

The synthesis of 7-ACA from cephalosporin C (CPC) by a two-step bioconversion using D-amino acid oxidase (DAAO) and glutaryl 7-ACA acylase (GLA) has been effectively and largely applied in pharmaceutical industry. In this study, the gene gla coding for 720-amino acid GLA from plasmid pUC57::gla was analyzed and successfully inserted into vector pET22b(+) to form expression vector pET22b(+)::gla. The newly constructed expression vector pET22b(+)::gla was cloned and then transformed into Escherichia coli BL21(DE3) to generate recombinant strain E. coli BL21(DE3)[pET22b(+)::gla]. The suitable conditions for expression of gla gene were in LB medium at 30 oC and induced by 0.4 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours. Under the chosen culturing parameters, expression of gla gene by E. coli BL21(DE3)/[pET22b(+)::gla] resulted in a recombinant GLA (rGLA) with molecular weight of 83 kDa and catalytic activity of 2.7 U/mg of total protein. Experimental research on immobilization of rGLA onto ten nanoporous materials were showed that, SBA-15 was the best one for immobilization of rGLA, reaching activity of immobilized enzyme of 22.2 U/g matrix. Furthermore, optimal conditions of procedure for immobilizing rGLA on nanomaterials (SBA-15) were determined as follows: temperature is 25 °C, pH7.0 and immobilization time –60 minutes. Therefore the results reported in this study revealed the successfully heterologous expression of GLA in recombinant E. coli and potential immobilization of enzyme on inorganic nano-materials.

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