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 Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1): structure–function relationships of RNA editing and dimerization

1999 ◽  
Vol 40 (4) ◽  
pp. 623-635 ◽  
Author(s):  
Ba-Bie Teng ◽  
Scott Ochsner ◽  
Qian Zhang ◽  
Kizhake V. Soman ◽  
Paul P. Lau ◽  
...  
Keyword(s):  
Structure Function ◽  
Rna Editing ◽  
Apolipoprotein B ◽  
Mutational Analysis ◽  
Mrna Editing ◽  
Editing Enzyme

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

scholarly journals Purification and Molecular Cloning of a Novel Essential Component of the Apolipoprotein B mRNA Editing Enzyme-Complex

2000 ◽  
Vol 275 (26) ◽  
pp. 19848-19856 ◽  
Author(s):  
Heinrich Lellek ◽  
Romy Kirsten ◽  
Ines Diehl ◽  
Frank Apostel ◽  
Friedrich Buck ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Apolipoprotein B ◽  
Enzyme Complex ◽  
Mrna Editing ◽  
Essential Component ◽  
Editing Enzyme

scholarly journals Apolipoprotein B mRNA–Editing Enzyme, Catalytic Polypeptide–Like 3G: A Possible Role in the Resistance to HIV of HIV‐Exposed Seronegative Individuals

2007 ◽  
Vol 195 (7) ◽  
pp. 960-964 ◽  
Author(s):  
Mara Biasin ◽  
Luca Piacentini ◽  
Sergio Lo Caputo ◽  
Yasuyoshi Kanari ◽  
Giuliana Magri ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Mrna Editing ◽  
Hiv Exposed ◽  
Editing Enzyme

Assignment of the Gene Encoding the Human Apolipoprotein B mRNA Editing Enzyme (APOBEC1) to Chromosome 12p13.1

Genomics ◽  
1994 ◽  
Vol 24 (2) ◽  
pp. 414-415 ◽  
Author(s):  
Rafael Espinosa ◽  
Toru Funahashi ◽  
Christos Hadjiagapiou ◽  
Michelle M. Le Beau ◽  
Nicholas O. Davidson
Keyword(s):  
Apolipoprotein B ◽  
Mrna Editing ◽  
Gene Encoding ◽  
Human Apolipoprotein ◽  
Editing Enzyme

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 Correlation of Apolipoprotein B mRNA-editing Enzyme, Catalytic Polypeptide- like 3G Genetic Variant rs8177832 with HIV-1 Predisposition in Pakistani Population

2019 ◽  
Vol 16 (4) ◽  
pp. 297-301
Author(s):  
Khurshid Iqbal ◽  
Muhammad Imran ◽  
Shafi Ullah ◽  
Muhsin Jamal ◽  
Yasir Waheed
Keyword(s):  
Genetic Variation ◽  
Apolipoprotein B ◽  
Host Factors ◽  
Host Susceptibility ◽  
Mrna Editing ◽  
Pakistani Population ◽  
Healthy Control ◽  
Important Host ◽  
Editing Enzyme ◽  
Hiv 1

Background: Human immunodeficiency virus (HIV) infection is a global health burden which ultimately results in acquired immune deficiency syndrome (AIDS). There are multiple host factors which are capable of limiting HIV-1 replication. One of the most important host factors which inhibit HIV-1 DNA synthesis is the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide- like 3G (APOBEC3G). Any genetic variation of this important host factor may influence the host susceptibility to viral infection. Objective: The aim of the current study was to evaluate any correlation of APOBEC3G genetic variation rs8177832 with HIV-1 infection. Methods: The study involved 142 healthy control and 100 HIV-1 infected subjects. The genetic variation rs8177832 of all studied subjects was determined by allele-specific polymerase chain reaction (AS-PCR). Results: The results showed that the distribution of rs8177832 genotypes AA, AG and GG in healthy subjects and HIV-1 subjects was; 42.253%, 42.957%, 14.788% and 66%, 27%, 7% respectively. Statistical analyses of data showed that there was a significant variation in rs8177832 genotype AA in healthy control and HIV-1 infected subjects (42.257% vs 66%; p-value<0.001). Conclusion: Thus it was concluded that APOBEC3G rs8177832 AA genotype contributes in genetic predisposition to HIV-1 infection in Pakistani population.

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