scholarly journals Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

2004 ◽  
Vol 384 (2) ◽  
pp. 385-390 ◽  
Author(s):  
Vesa P. HYTÖNEN ◽  
Olli H. LAITINEN ◽  
Tomi T. AIRENNE ◽  
Heidi KIDRON ◽  
Niko J. MELTOLA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Specific Binding ◽  
Affinity Purification ◽  
Active Form ◽  
Efficient Production ◽  
E Coli ◽  
Secretion Signal ◽  
Technology Applications ◽  
Biotin Binding

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin–agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380–1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin–biotin technology applications.

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

scholarly journals Lectin and E. coli Binding to Carbohydrate-Functionalized Oligo(ethylene glycol)-Based Microgels: Effect of Elastic Modulus, Crosslinker and Carbohydrate Density

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 263
Author(s):  
Fabian Schröer ◽  
Tanja J. Paul ◽  
Dimitri Wilms ◽  
Torben H. Saatkamp ◽  
Nicholas Jäck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Elastic Modulus ◽  
Ethylene Glycol ◽  
Concanavalin A ◽  
Specific Binding ◽  
Network Density ◽  
Carbohydrate Binding ◽  
E Coli ◽  
Surface Recognition ◽  
Strong Anchoring

The synthesis of carbohydrate-functionalized biocompatible poly(oligo(ethylene glycol) methacrylate microgels and the analysis of the specific binding to concanavalin A (ConA) and Escherichia coli (E. coli) is shown. By using different crosslinkers, the microgels’ size, density and elastic modulus were varied. Given similar mannose (Man) functionalization degrees, the softer microgels show increased ConA uptake, possibly due to increased ConA diffusion in the less dense microgel network. Furthermore, although the microgels did not form clusters with E. coli in solution, surfaces coated with mannose-functionalized microgels are shown to bind the bacteria whereas galactose (Gal) and unfunctionalized microgels show no binding. While ConA binding depends on the overall microgels’ density and Man functionalization degree, E. coli binding to microgels’ surfaces appears to be largely unresponsive to changes of these parameters, indicating a rather promiscuous surface recognition and sufficiently strong anchoring to few surface-exposed Man units. Overall, these results indicate that carbohydrate-functionalized biocompatible oligo(ethylene glycol)-based microgels are able to immobilize carbohydrate binding pathogens specifically and that the binding of free lectins can be controlled by the network density.

scholarly journals Thermal inactivation and chaperonin-mediated renaturation of mitochondrial aspartate aminotransferase

1998 ◽  
Vol 334 (1) ◽  
pp. 219-224 ◽  
Author(s):  
James M. LAWTON ◽  
Shawn DOONAN
Keyword(s):  
Escherichia Coli ◽  
Aspartate Aminotransferase ◽  
Thermal Inactivation ◽  
Active Form ◽  
Active Conformation ◽  
E Coli ◽  
Catalytically Active ◽  
Mitochondrial Aspartate Aminotransferase ◽  
Chaperonin 10 ◽  
Chaperonin 60

Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55–65 °C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37–44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55–60 °C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.

scholarly journals Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material

2020 ◽  
Vol 47 (12) ◽  
pp. 1117-1132
Author(s):  
Katharina Novak ◽  
Juliane Baar ◽  
Philipp Freitag ◽  
Stefan Pflügl
Keyword(s):  
Escherichia Coli ◽  
Cheese Whey ◽  
Strain Improvement ◽  
Defined Medium ◽  
Raw Material ◽  
Efficient Production ◽  
Substrate Uptake ◽  
Chemically Defined Medium ◽  
E Coli ◽  
Plasmid Library

AbstractThe aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l−1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes.

scholarly journals Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4891-4896 ◽  
Author(s):  
Ji Qiu ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Disulfide Bonds ◽  
Specific Activity ◽  
Active Form ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
E Coli ◽  
Disulfide Isomerases

ABSTRACT The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coli without the need for in vitro refolding.

scholarly journals Efficient production of (S)-1-phenyl-1, 2-ethanediol using xylan as co-substrate by a coupled multi-enzyme Escherichia coli system

2020 ◽  
Author(s):  
Junchao Rao ◽  
Rongzhen Zhang ◽  
Guanyu Xu ◽  
Lihong Li ◽  
Yan Xu
Keyword(s):  
Escherichia Coli ◽  
Glucose Dehydrogenase ◽  
Optical Purity ◽  
Carbonyl Reductase ◽  
Cofactor Regeneration ◽  
Efficient Production ◽  
Chiral Synthesis ◽  
E Coli ◽  
Concomitant Reduction ◽  
Different Strains

Abstract Background: ( S )-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines.(S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to ( S )-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP + to NADPH, while endo-β-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 were introduced into the ( S )-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. Results: We constructed several coupled multi-enzyme systems by introducing ( S )-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 into Escherichia coli . Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli /pET-G-S-2 expressed all three enzymes, and this strain produced ( S )-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35°C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-β-1,4-xylanase 2 into the ( S )-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 h to 28 h. Conclusions: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.

scholarly journals Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Morphological Changes ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Prolonged Incubation ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

scholarly journals Anti-Escherichia coli Functionalized Silver-Doped Carbon Nanofibers for Capture of E. coli in Microfluidic Systems

Polymers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1117 ◽  
Author(s):  
Soshana Smith ◽  
Michael Delaney ◽  
Margaret Frey
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Carbon Nanofibers ◽  
Specific Binding ◽  
Base Material ◽  
Fiber Surface ◽  
Electrospinning Process ◽  
Silver Particles ◽  
Microfluidic Systems ◽  
E Coli

Silver-doped carbon nanofibers (SDCNF) are used as the base material for the selective capture of Escherichia coli in microfluidic systems. Fibers were spun in a glovebox with dry atmosphere maintained by forced dry air pumped through the closed environment. This affected the evaporation rate of the solvent during the electrospinning process and the distribution of silver particles within the fiber. Antibodies are immobilized on the surface of the silver-doped polyacrylonitrile (PAN) based carbon nanofibers via a three-step process. The negatively charged silver particles present on the surface of the nanofibers provide suitable sites for positively charged biotinylated poly-(L)-lysine-graft-poly-ethylene-glycol (PLL-g-PEG biotin) conjugate attachment. Streptavidin and a biotinylated anti-E. coli antibody were then added to create anti-E. coli surface functionalized (AESF) nanofibers. Functionalized fibers were able to immobilize up to 130 times the amount of E. coli on the fiber surface compared to neat silver doped fibers. Confocal images show E. coli remains immobilized on fiber mat surface after extensive rinsing showing the bacteria is not simply a result of non-specific binding. To demonstrate selectivity and functionalization with both gram negative and gram-positive antibodies, anti-Staphylococcus aureus surface functionalized (ASSF) nanofibers were also prepared. Experiments with AESF performed with Staphylococcus aureus (S. aureus) and ASSF with E. coli show negligible binding to the fiber surface showing the selectivity of the functionalized membranes. This surface functionalization can be done with a variety of antibodies for tunable selective pathogen capture.

scholarly journals Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

scholarly journals Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975

1999 ◽  
Vol 341 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Donna D. SONG ◽  
Nicholas A. JACQUES
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Streptococcus Salivarius ◽  
Acetobacter Diazotrophicus ◽  
E Coli ◽  
Ping Pong ◽  
Mechanism Of Catalysis ◽  
Enzyme Intermediate

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 °C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.

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