Effect of consecutive rare codons on the recombinant production of human proteins in <i>Escherichia coli</i>

Correddu, Danilo; López, José de Jesús Montaño; Angermayr, S. Andreas; Middleditch, Martin; Payne, Leo S.; Leung, Ivanhoe K. H.

doi:10.1002/iub.2162

Cited by 6 publications

(3 citation statements)

References 19 publications

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“…Since the only difference between the native phaB and phaB _L are two nucleotides (138-CAG and 130-CTG, respectively) and the same two nucleotides were altered to 138-CAA in phaB -opt ( Supplementary Figure 5 ) it can be assumed that the deleterious effect stems from translational complications in this region. Correddu et al (2019) found that co-occurrence of certain amino acid codons in E. coli led to a premature stop in the protein chain potentially through a frameshift. Similar effects could be at play here.…”

Section: Discussionmentioning

confidence: 99%

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

Grosse-Honebrink

Little

Bean

et al. 2021

Front. Bioeng. Biotechnol.

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Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.

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Section: Discussionmentioning

confidence: 99%

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

Grosse-Honebrink

Little

Bean

et al. 2021

Front. Bioeng. Biotechnol.

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“…Persentase kandungan GC dari gen hasil optimasi meningkat menjadi 56,57% dan berada dalam kisaran persentase ideal yaitu 30-70% (GenScript, 2019). Correddu et al, (2019) melaporkan translasi yang disebabkan oleh kodon arginin langka (AGG-AGA) pada E. coli, menghasilkan produksi protein RioK2 terpotong. Penggantian kodon langka AGG-AGA dengan kodon arginine CGC-CGT yang dikenali oleh E. coli sebagai inang mencegah produksi protein terpotong.…”

Section: Optimasi Gen Penyandi Lon-like Proteaseunclassified

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

et al. 2019

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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.

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“…Recombinant DNA technology has made it possible to produce bulk quantities of enzymes and proteins of pharmaceutical importance in E. coli and Bacillus species. Genetically modified bacterial strains with disrupted native protease and RNase enzymes, preferred tRNA genes and genome having lysogenically introduced bacteriophage T7 RNA polymerase gene are often used as a tool for recombinant protein production (Gopal and Kumar, 2013;Correddu et al, 2020;Duarte et al, 2020;Doozandeh-Juibari et al, 2020). Pyrococcus abyssi is an archaeon that grows under stringent conditions with optimum growth temperature 96°C, under anaerobic high pressure conditions (Erauso et al, 1993), its genome has been analysed (Cohen et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

et al. 2022

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L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G – 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

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Effect of consecutive rare codons on the recombinant production of human proteins in Escherichia coli

Cited by 6 publications

References 19 publications

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol

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

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

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