Growth Inhibition of Human Melanoma Cells by a Recombinant Arginine Deiminase Expressed in Escherichia coli

Kozai, Megumi; Sasamori, Eriko; Fujihara, Masatoshi; Yamashita, Tetsuro; Taira, Hideharu; Harasawa, Ryô

doi:10.1292/jvms.001343

Cited by 12 publications

(6 citation statements)

References 26 publications

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“…Here, PAL was shown to be toxic for breast cancer MCF7 and prostate cancer DU145 cells. Compared with other anticancer enzymes, PAL has the similar cytotoxic effect with l ‐asparaginases and novel anticancer enzymes such as arginine deiminase and onconase . For example, IC 50 of l ‐asparaginase from E .…”

Section: Discussionmentioning

confidence: 99%

Recombinant l‐phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent

Babich

Pokrovsky²,

Anisimova

et al. 2013

Biotech and App Biochem

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The recombinant producer strain expressing Rhodosporidium toruloides l-phenylalanine ammonia lyase (PAL) has been obtained, and a purification procedure of PAL has been developed. The purified enzyme, PAL, has the following biochemical and catalytic characteristics: Km for l-Phe of 0.49 mM, pH optimum at 8.5, and temperature optimum at 50°C. PAL exhibited a significant cytotoxic effect toward the following cell lines: MCF7 (IC50 = 1.97 U/mL), DU145 (IC50 = 7.3 U/mL), which are comparable with E. coli l-asparaginase type-II cytotoxicity in vitro. Administration of PAL (200-400 U/kg) to L5178y-bearing mice for five times (a total dose of 1000-2000 U/kg) was well tolerated and showed the increase of life span (ILS) = 12-16%, P < 0.05. Data obtained suggest that PAL from R. toruloides has a potential for cancer treatment.

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Section: Discussionmentioning

confidence: 99%

Recombinant l‐phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent

Babich

Pokrovsky²,

Anisimova

et al. 2013

Biotech and App Biochem

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“…[30] reported that the addition of L-arginine in the refolding solution was able to clearly increase the enzymatic activity of denatured ADI proteins. However, our experiments showed that the presence of L-arginine had no effect on the in-vitro solubilization of rADI proteins in the inclusion body form.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, it had been observed that the improvement of the substrate L-arginine for correct refolding of denatured ADI proteins in the refolding solution [30]. However, L-arginine had no effect on the in vitro solubilization of rADI proteins in inclusion body forms.…”

Section: Discussionmentioning

confidence: 99%

Cultivation to improve in vivo solubility of overexpressed arginine deiminases in Escherichia coliand the enzyme characteristics

Wang

2014

BMC Biotechnol

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BackgroundOverexpression of foreign genes in Escherichia coli cells is an efficient means to obtain recombinant proteins. The technique is, however, often hampered by misfolding, degradation, aggregation and formation in inclusion bodies of products.ResultsIn this study, we reported that in vivo solubility of overexpressed arginine deiminases (ADI) improved by changing the cultivation conditions. ADI is enzymes that convert L-arginine to L-citrulline. After codon optimization, we synthesized the ADI gene of Pseudomonas putida and constructed it for overexpression in E. coli cells. The rADI products were mainly in inclusion body forms. We performed a series of optimization to enhance solubility of the protein. Co-expression with the GroES-GroEL chaperone team increased approximately 5-fold of the rADI activity. In addition the combination of L-arginine and D-glucose in the Luria-Bertani (LB) growth medium further increased the total activity to about 15 times. Separate L-arginine and D-glucose or the addition of other saccharides or amino acids had no such effects. The solubilization effects of the combination of L-arginine and D-glucose were further confirmed in the overexpression of another ADI from Listeria welshimeri. The enzymatic and conversion characteristics of the rADI products were further determined.ConclusionsCombined addition of L-arginine and D-glucose in the LB medium significantly improved in vivo solubility of rADI proteins. The present study suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in E. coli cells.

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“…The inhibitory effect of native or recombinant arginine deiminase on the proliferation of tumor cells makes this enzyme a candidate for antitumor treatment (45). The main effects of arginine deiminase on these cells are depletion of arginine (46), inhibition of the cell cycle, and induction of apoptosis (41).…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of an Active Arginine Deiminase Pathway in Mycoplasma pneumoniae M129

Rechnitzer¹,

Rottem

Herrmann

2013

Infect Immun

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b Some species of the genus Mycoplasma code for the arginine deiminase pathway (ADI), which enables these bacteria to produce ATP from arginine by the successive reaction of three enzymes: arginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB), and carbamate kinase (ArcC). It so far appears that independently isolated strains of Mycoplasma pneumoniae encode an almost identical truncated version of the ADI pathway in which the proteins ArcA and ArcB have lost their original enzymatic activities due to the deletion of significant regions of these proteins. To study the consequences of a functional ADI pathway, M. pneumoniae M129 was successfully transformed with the cloned functional arcA, arcB, and arcC genes from Mycoplasma fermentans. Enzymatic tests showed that while the M. pneumoniae ArcAB and ArcABC transformants possess functional arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase, they were unable to grow on arginine as the sole energy source. Nevertheless, infection of a lung epithelial cell line, A549, with the M. pneumoniae transformants showed that almost 100% of the infected host cells were nonviable, while most of the lung cells infected with nontransformed M. pneumoniae were viable under the same experimental conditions. T he mycoplasmas (class Mollicutes) form a large group of prokaryotic microorganisms that are divided into nine genera with over 200 species. They are distinguished from ordinary bacteria by their small size and minute genome (0.58 to 2.20 Mb) and the total lack of a cell wall (1, 2). Phylogenetically, the mycoplasmas are related to Gram-positive bacteria, from which they developed by genome reduction (3).One criterion for classifying and characterizing mycoplasma species has been their energy source. For instance, glycolytic mycoplasmas generate energy from sugars by glycolysis, while some of the nonglycolytic mycoplasmas catabolize arginine by the arginine deiminase (ADI) pathway, consisting of three enzymes: arginine deiminase (ArcA), which hydrolyzes arginine to citrulline and ammonia; ornithine carbamoyltransferase (ArcB), which converts citrulline in the presence of phosphate to ornithine and carbamoylphosphate; and carbamate kinase (ArcC), which synthesizes ATP from carbamoylphosphate and ADP. For clarity and simplicity, throughout this article these protein and gene names are interchangeable.The presence of glycolytic and/or ADI pathways in bacteria has been analyzed in the past by selective growth conditions and enzymatic assays. Barile and coworkers (4) tested 18 Mycoplasma species, of which 10, including Mycoplasma hominis, M. arthriditis, and M. fermentans, could convert arginine to ATP by the arginine deiminase pathway. Among the ADI-negative Mycoplasma species was M. pneumoniae M129, whose complete genome has been sequenced and annotated (5). Surprisingly, an operon that contains coding DNA sequences (CDSs) with significant similarities to all three enzymes of the ADI pathway and a hypothetical arginine transporter have been described fo...

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Growth Inhibition of Human Melanoma Cells by a Recombinant Arginine Deiminase Expressed in Escherichia coli

Cited by 12 publications

References 26 publications

Recombinant l‐phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent

Recombinant l‐phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent

Cultivation to improve in vivo solubility of overexpressed arginine deiminases in Escherichia coliand the enzyme characteristics

Reconstitution of an Active Arginine Deiminase Pathway in Mycoplasma pneumoniae M129

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