Optimization of the Fusion Expression of Chicken β-Defensin Gal-3 in E Coli

2013 ◽  
Vol 634-638 ◽  
pp. 1343-1346
Author(s):  
Hui Yan Wang ◽  
Lei Liu ◽  
Suo Mo ◽  
Huan Liu ◽  
Yuan Xin He ◽  
...  
Keyword(s):  
Recombinant Strain ◽  
Final Concentration ◽  
Optimal Parameters ◽  
Culture Temperature ◽  
Iptg Induction ◽  
Initial Cell ◽  
E Coli ◽  
Lactose Induction ◽  
Lactose Concentration ◽  
Large Production

The expression level of chicken β-defensin Gal-3 was influenced by lactose concentration, induction time and initial cell density, and IPTG induction was used as a control. The optimal parameters of various factors were confirmed. the optimal parameters with lactose induction was 37°C of culture temperature, 2 g/L of joining lactose final concentration when the recombinant strain growth density A600 achieved 0.8 and induction maintained 3 hours, the production of fusion protein reached the highest level. This study provides a good experimental basis for the large production of Gal-3.

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

1998 ◽  
Vol 64 (3) ◽  
pp. 1163-1165 ◽  
Author(s):  
Georges Feller ◽  
Olivier Le Bussy ◽  
Charles Gerday
Keyword(s):  
Escherichia Coli ◽  
Enzyme Stability ◽  
Expression System ◽  
Wild Strain ◽  
Culture Temperature ◽  
E Coli ◽  
System A ◽  
Irreversible Denaturation ◽  
The Antarctic ◽  
Folding State

ABSTRACT α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.

scholarly journals Optimization of the apolipoprotein B mRNA editing enzyme catalytic polypeptidelike-3G (APOBEC3G) gene to enhance its expression in Escherichia coli

2020 ◽  
Vol 29 (2) ◽  
pp. 120-8
Author(s):  
Rizkyana Avissa ◽  
Silvia Tri Widyaningtyas ◽  
Budiman Bela
Keyword(s):  
Escherichia Coli ◽  
Apolipoprotein B ◽  
Plasmid Vector ◽  
Nitrilotriacetic Acid ◽  
Mrna Editing ◽  
Specific Domain ◽  
Iptg Induction ◽  
E Coli ◽  
Gene Coding ◽  
Editing Enzyme

BACKGROUND Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like-3G (APOBEC3G) can abolish HIV infection by inducing lethal mutations in the HIV genome. The HIV protein virion infectivity factor (Vif) can interact with APOBEC3G protein and cause its degradation. Development of a method that can screen substances inhibiting the APOBEC3G-Vif interaction is necessary for identification of substances that potentially used in anti-HIV drug development. In order to increase expression of recombinant APOBEC3G protein that will be used in APOBEC3G-Vif interaction assay, we developed an optimized APOBEC3G gene for expression in Escherichia coli.  METHODS The gene coding APOBEC3G was codon-optimized in accordance with prokaryotic codon using DNA 2.0 software to avoid bias codons that could inhibit its expression. The APOBEC3G gene was synthesized and sub-cloned into pQE80L plasmid vector. pQE80L containing APOBEC3G was screened by polymerase chain reaction, enzyme restriction, and sequencing to verify its DNA sequence. The recombinant APOBEC3G was expressed in E. coli under isopropyl-β-D-thiogalactoside (IPTG) induction and purified by using nickel-nitrilotriacetic acid (Ni-NTA) resin.  RESULTS The synthetic gene coding APOBEC3G was successfully cloned into the pQE80L vector and could be expressed abundantly in E. coli BL21 in the presence of IPTG.  CONCLUSIONS Recombinant APOBEC3G is robustly expressed in E. coli BL21, and the APOBEC3G protein could be purified by using Ni-NTA. The molecular weight of the recombinant APOBEC3G produced is smaller than the expected value. However, the protein is predicted to be able to interact with Vif because this interaction is determined by a specific domain located on the N-terminal of APOBEC3G. 

scholarly journals Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

2019 ◽  
Vol 19 (2) ◽  
pp. 149-158
Author(s):  
Olfa Mega ◽  
Cece Sumantri ◽  
Irma Isnafia Arief ◽  
Cahyo Budiman
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Plantarum ◽  
Expression System ◽  
Gc Content ◽  
Whole Genome Sequence ◽  
Protease Gene ◽  
Iptg Induction ◽  
E Coli ◽  
Sds Page ◽  
Index Value

Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico.  pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively.  Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C.  As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.

scholarly journals Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Qin Fan ◽  
Peter Neubauer ◽  
Matthias Gimpel
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Protein Production ◽  
Ralstonia Eutropha ◽  
Process Time ◽  
Synthesis Rate ◽  
Heterologous Production ◽  
Fed Batch ◽  
Iptg Induction ◽  
E Coli

Abstract Background Autoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate. Results We were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date. Conclusions The findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.

scholarly journals A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)

2020 ◽  
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Kazuyoshi Hirota ◽  
Shirou Itagaki
Keyword(s):  
Topoisomerase Ii ◽  
Fluorescent Proteins ◽  
Expression System ◽  
Simple Expression ◽  
Amino Acid Sequences ◽  
Expression Plasmid ◽  
Iptg Induction ◽  
E Coli ◽  
Mammalian Expression ◽  
Effective Selection

AbstractWe introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. III EVOLUTION OF THE ebg REPRESSOR DURING SELECTION FOR ENHANCED LACTASE ACTIVITY

Genetics ◽  
1977 ◽  
Vol 85 (2) ◽  
pp. 193-201
Author(s):  
Barry G Hall ◽  
Norma D Clarke
Keyword(s):  
Regulatory Gene ◽  
Enzyme Synthesis ◽  
Lactase Activity ◽  
Wild Type ◽  
Regulatory Mutation ◽  
E Coli ◽  
Lactose Induction ◽  
A Cell ◽  
Lactose Utilization ◽  
Selection For

ABSTRACT The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg  + strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.

scholarly journals Improving vaccine efficacy based on non-covalent photoinactivation of microorganisms - sources vaccine antigens (MSVA)

2020 ◽  
Author(s):  
Artur Martynov ◽  
Boris Farber ◽  
Tatyana Osolodchenko ◽  
Ilya Klein
Keyword(s):  
Protective Effect ◽  
Blue Light ◽  
Bacterial Growth ◽  
Final Concentration ◽  
Control Group ◽  
Photodynamic Inactivation ◽  
E Coli ◽  
Light Emitter ◽  
Vaccine Antigens ◽  
Inactivated Vaccines

AbstractOne of the most promising methods for non-covalent inactivation of vaccine-producing microorganisms is the use of photoinactivation using riboflavin derivatives. The study used a dynamic combinatorial derivative of riboflavin - succinyl-maleinyl riboflavin. Corpuscular vaccines are divided into the following groups: from 2AB to 5AB - bacteria were inactivated by riboflavin derivative and blue light, and groups from 6AB to 9AB were inactivated by formalin (0.1% formalin in 9 log CFU was kept for 2 weeks in an thermostat and then sterility was determined - bacterial growth was not observed). A dynamic derivative of riboflavin at a final concentration of 0.02% can photo inactivate 6 time more bacteria P. Aeruginosa and E. coli than riboflavin. The minimum effective blue light emitter power (450 nm) for the photodynamic inactivation of both P. aeruginosa and E. coli is 1024.2 mW / cm2. In groups of mice pre-vaccinated intraperitoneally with corpuscular photo inactivated vaccines based on suspensions of and E. coli at doses of 0.5-1.0 ml 4 log (CFU) / mL, 100% survival of all animals was observed, whereas in control group with the same type of vaccines but formalin-treated vaccines, it failed to achieve a 100% protective effect.

Repressive effects of yeast extract on photoreactivation of Escherichia coli

2004 ◽  
Vol 50 (1) ◽  
pp. 33-38
Author(s):  
J. Oguma ◽  
H. Katayama ◽  
H. Mitani ◽  
S. Ohgaki
Keyword(s):  
Escherichia Coli ◽  
Yeast Extract ◽  
Final Concentration ◽  
Survival Ratio ◽  
E Coli ◽  
Molecular Weights ◽  
Pyrimidine Dimers ◽  
Sensitive Site ◽  
Uv Lamp ◽  
Nominal Molecular Weight

Photoreactivation of Escherichia coli K12 (IFO 3301), in the presence or absence of yeast extract (YE), was investigated after inactivation by low-pressure UV lamp. An endonuclease sensitive site (ESS) assay was used to determine the UV-induced pyrimidine dimers in the genome of E. coli, while a colony-forming ability (CFA) test was also used to examine the survival ratio of E. coli. The YE solution reduced the CFA recovery at a final concentration of 125 mg/L. A dialysis of the YE solution indicated that the YE fraction (with nominal molecular weight >1,000 and <3,500) was effective at repressing the CFA recovery. Interestingly, the repair of ESS was equivalent regardless of the presence of the YE dialysate, while the CFA recovery was significantly repressed in the presence of YE. It was, therefore, suggested that YE components, probably with molecular weights of 1,000-3,500, were effective at repressing the CFA recovery of E. coli without affecting the ESS repair during photoreactivation.

scholarly journals The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

2020 ◽  
Vol 7 (1) ◽  
pp. 8 ◽  
Author(s):  
Johanna Hausjell ◽  
Regina Kutscha ◽  
Jeannine D. Gesson ◽  
Daniela Reinisch ◽  
Oliver Spadiut
Keyword(s):  
Expression System ◽  
Stable Process ◽  
Product Formation ◽  
Lac Repressor ◽  
Process Conditions ◽  
Expression Systems ◽  
Iptg Induction ◽  
Lactose Induction ◽  
A Genome ◽  
Uptake Rates

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

scholarly journals Inhibitory Potential Of Lactobacillus Plantarum on Escherichia Coli

2017 ◽  
Vol 74 (2) ◽  
pp. 99 ◽  
Author(s):  
Laura MITREA ◽  
Lavinia Florina CĂLINOIU ◽  
Gabriela PRECUP ◽  
Maria BINDEA ◽  
Bogdan RUSU ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Plantarum ◽  
Alternative Therapy ◽  
Optimal Parameters ◽  
E Coli ◽  
Enteropathogenic Bacteria ◽  
Probiotic Strains ◽  
Inhibitory Potential ◽  
Mrs Broth

An alternative therapy to diarrhoeal episodes induced by E. coli is represented by probiotic strains (e.g Lactobacillus), which are able to maintain the gut micro-flora in optimal parameters. This work highlights the inhibitory potential of probiotics (L. plantarum ATCC 8014) over the enteropathogenic bacteria like E. coli ATCC 25922. For this study we co-incubated L. plantarum with E. coli in MRS broth at different concentrations (109, 108 CFU/mL – L. plantarum with 105, 104 CFU/mL – E. coli, 4 groups) for 24, 48 and 72h at 37oC. After co-incubation, E. coli colonies developed on Levine media were counted towards the control. E. coli colonies decreased after 48 and 72h of co-incubation with L. plantarum. Also, was noticed that L. plantarum (108 CFU/mL) completely inhibits E. coli (104 CFU/mL) after 72h of co-incubation. This work proves the inhibitory potential of probiotic strains (L. plantarum ATCC 8014) against enteropathogenic bacteria (E. coli ATCC 25922).

Sign in / Sign up

Export Citation Format

Share Document