Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study

Burgess-Brown, N.; Sharma, Sujata; Sobott, Frank; Loenarz, Christoph; Oppermann, U.; Gileadi, O.

doi:10.1016/j.pep.2008.01.008

Cited by 288 publications

(203 citation statements)

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“…The Blh protein was expressed as a soluble form in E. coli, and the protein was purified without detergent even though it is a member of the bacteriorhodopsin family. Its soluble form may result from the codon optimization, which is known to increase the expression and solubility of enzymes (39).…”

Section: Resultsmentioning

confidence: 99%

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

Kim

Yeom

et al. 2009

Journal of Biological Chemistry

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Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These ␤-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).␤-Carotene 15,15Ј-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of ␤-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from ␤-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of ␤-carotene 15,15Ј-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert ␤-carotene to retinal include bacterioopsin-r...

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Section: Resultsmentioning

confidence: 99%

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

Kim

Yeom

et al. 2009

Journal of Biological Chemistry

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“…The IL6(T23)-PE38KDEL gene using E. coli preferred codons was designed to overcome the bottleneck caused by the low expression level of the natural gene in the pET expression system. Codon optimization has become a very effective means to improve protein expression (24,31,32). In a previous study, we constructed the IL6(T23)-PE38KDEL gene by overlap PCR using the natural IL6 and PE genes.…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo antitumor effects of the recombinant immunotoxin IL6(T23)-PE38KDEL in multiple myeloma

Guo

Han

et al. 2012

Oncology Letters

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Abstract. IL6(T23)-PE38KDEL is a chimeric molecule composed of interleukin 6 (IL6), missing the N-terminal 23 amino acids, and fused to a truncated mutant form of Pseudomonas exotoxin (PE38KDEL). The aim of this study was to evaluate this recombinant immunotoxin in terms of its specific cytotoxicity to IL6R-overexpressing multiple myeloma (MM) cells in vitro, as well as its antitumor effects and side effects in vivo. IL6(T23)-PE38KDEL was expressed in Escherichia coli, refolded and purified from inclusion bodies. The purified IL6(T23)-PE38KDEL was found to be selectively cytotoxic to IL6 receptor-positive tumor cells in vitro. IC 50 values of IL6(T23)-PE38KDEL were evaluated by MTS assay. Toxicity and maximum-tolerated dose of IL6(T23)-PE38KDEL were determined in mice. The antitumor activity of IL6(T23)-PE38KDEL was evaluated in mice with MM through intravenous injection and interventional therapy. Intravenous administration of IL6(T23)-PE38KDEL caused a significantly increased survival time in treated mice, and exhibited dose-and time-dependent antitumor effects against MM mice. Moreover, complete tumor regression was observed in 30 and 80% of mice treated intravenously and intraperitoneally, respectively, with 0.4 mg/kg/day for 10 days. These results demonstrated that the recombinant immunotoxin IL6(T23)-PE38KDEL kills IL6R-overexpressing cancer cells, and causes significant tumor regression.

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“…Organisms have developed individual preferences for particular codons for a given amino acid (known as 'codon usage bias'). As the efficient generation of PVs requires good levels of VEP expression in the producer cells, codon optimization relevant for the given producer cell line may improve viral titers [108][109][110][111].…”

Section: Influence Of Plasmid and Codon Optimizationmentioning

confidence: 99%

Technical Considerations for the Generation of Novel Pseudotyped Viruses

et al. 2015

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Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study

Cited by 288 publications

References 32 publications

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

In vitro and in vivo antitumor effects of the recombinant immunotoxin IL6(T23)-PE38KDEL in multiple myeloma

Technical Considerations for the Generation of Novel Pseudotyped Viruses

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