scholarly journals A Generic, Whole-Cell–Based Screening Method for Baeyer-Villiger Monooxygenases

2013 ◽  
Vol 18 (6) ◽  
pp. 678-687 ◽  
Author(s):  
Hanna M. Dudek ◽  
Petra Popken ◽  
Edwin van Bloois ◽  
Wouter A. Duetz ◽  
Marco W. Fraaije
Keyword(s):  
Detection Method ◽  
Catalytic Properties ◽  
Expression System ◽  
Screening Method ◽  
Whole Cell ◽  
E Coli ◽  
Coenzyme Regeneration ◽  
Escherichia Coli Cells ◽  
Phosphite Dehydrogenase ◽  
Screening Site

Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell–based screening method was validated by screening site–saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.

scholarly journals Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

2003 ◽  
Vol 50 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Anna-Maria Ochocka ◽  
Marzena Czyzewska ◽  
Tadeusz Pawełczyk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Rho Gtpase ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Step Procedure ◽  
Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

scholarly journals Improvement of an Escherichia coli whole-cell biocatalyst for geranyl glucoside production using directed evolution

2021 ◽  
Author(s):  
Julian Ruediger ◽  
Wilfried Schwab
Keyword(s):  
Directed Evolution ◽  
Production Systems ◽  
Low Cost ◽  
Continuous Production ◽  
Expression System ◽  
Screening Method ◽  
Fold Increase ◽  
Small Scale ◽  
Biotechnological Production ◽  
Whole Cell

The biotechnological production of glycosides is an economically competitive manufacturing alternative to classical chemical synthesis. Through continuous production improvement, glycosides can now be used in low-cost products by various industries. However, many production systems still suffer from low yields. Directed evolution, coupled with a suitable screening method, can tackle this challenge. We generated glycosyltransferase mutants through error-prone-PCR and screened the library using a small-scale whole-cell biotransformation system. The screening of only 176 colonies yielded three putative candidates. Detailed investigations revealed that the reason for the increase in product titer was mainly due to different expression effects of the mutagenized genes rather than improved enzyme kinetics. In total, a 60-fold increase in product formation was achieved. Therefore, in addition to the quality of the mutant library, an efficient and stable expression system is crucial to achieve high concentrations of active enzyme and product, as formation of inclusion bodies and other inactive forms of the biocatalyst reduces productivity.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Aptamer-based SERS biosensor for whole cell analytical detection of E. coli O157:H7

2019 ◽  
Vol 1081 ◽  
pp. 146-156 ◽  
Author(s):  
Susana Díaz-Amaya ◽  
Li-Kai Lin ◽  
Amanda J. Deering ◽  
Lia A. Stanciu
Keyword(s):  
Whole Cell ◽  
E Coli ◽  
Analytical Detection

Viability and survival capability of quinolone-resistant uropathogenic Escherichia coli

2010 ◽  
Vol 5 (6) ◽  
pp. 827-830
Author(s):  
Georgi Slavchev ◽  
Nadya Markova
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Expression System ◽  
Antibiotic Sensitivity ◽  
Uropathogenic Escherichia Coli ◽  
Resistant Bacteria ◽  
E Coli ◽  
Serum Bactericidal Assay ◽  
Tract Infections ◽  
Macrophage Killing

AbstractUropathogenic strains of E. coli isolated from urine of patients with urinary tract infections were tested for antibiotic sensitivity using bio-Merieux kits and ATB-UR 5 expression system. The virulence of strains was evaluated by serum bactericidal assay, macrophage “killing” and bacterial adhesive tests. Survival capability of strains was assessed under starvation in saline. The results showed that quinolone-resistant uropathogenic strains of E. coli exhibit significantly reduced adhesive potential but relatively high resistance to serum and macrophage bactericidity. In contrast to laboratory strains, the quinolone-resistant uropathogenic clinical isolate demonstrated increased viability during starvation in saline. Our study suggests that quinolone-resistant uropathogenic strains are highly adaptable clones of E. coli, which can exhibit compensatory viability potential under unfavorable conditions. The clinical occurrence of such phenotypes is likely to contribute to the survival, persistence and spread strategy of resistant bacteria.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

Rapid E. coli-based cloning and expression system for production of recombinant proteins

2014 ◽  
Vol 185 ◽  
pp. S70
Author(s):  
Boguslaw Lupa ◽  
Krzysztof Stawujak ◽  
Igor Rozanski ◽  
Justyna Stec-Niemczyk
Keyword(s):  
Recombinant Proteins ◽  
Expression System ◽  
Cloning And Expression ◽  
E Coli

Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

An Improved Screening Method for the Detection and Isolation of Escherichia coli 0157:H7 From Meat, Incorporating the 3M Petrifilm™ Test Kit - HEC - for Hemorrhagic Escherichia coli 0157:H7

1990 ◽  
Vol 53 (11) ◽  
pp. 936-940 ◽  
Author(s):  
ANITA J. G. OKREND ◽  
BONNIE E. ROSE ◽  
RICHARD MATNER
Keyword(s):  
Escherichia Coli ◽  
Screening Program ◽  
Enrichment Culture ◽  
Screening Method ◽  
Meat Sample ◽  
E Coli ◽  
Enrichment Cultures ◽  
Poultry Products ◽  
Test Kit ◽  
Cooked Meat

A screening method was devised incorporating a commercially available reactive disc blot ELISA for Escherichia coli 0157 antigen, into a cultural screening program for the isolation of E. coli 0157:H7 from meat and poultry products. The method includes the inoculation of a raw or cooked meat sample into an enrichment broth, incubation with shaking at 37°C for 6 to 8 h, followed by inoculation of 3M Petrifilm™ E. coli Count plates with dilutions of the enrichment culture. The Petrifilm plates were incubated at 42°C for 18 h and tested for the presence of the 0157 antigen. The enrichment cultures were reincubated static at 35°C after the initial shaken incubation. Isolation was attempted from the positive Petrifilm plates by both a direct picking and streaking method and by the 3M Prompt™ isolation method. Isolation also was attempted from the 24-h enrichment cultures by spread plating serial dilutions on 150 × 15 mm MacConkey sorbitol agar (MSA) and MSA with 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid cyclohexylammonium salt (BCIG). This fast and efficient screening procedure identifies negative and presumptive positive samples in 26–28 h. Isolation and confirmation of the presumptive positive isolates require an additional 3 to 4 d.

scholarly journals Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Toxins ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 458 ◽  
Author(s):  
Hisaya Ono ◽  
Nobuaki Hachiya ◽  
Yasunori Suzuki ◽  
Ikunori Naito ◽  
Shouhei Hirose ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Expression System ◽  
Sandwich Elisa ◽  
Food Poisoning ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
E Coli ◽  
Detection Systems ◽  
C Terminus ◽  
Culture Supernatants

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

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