Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris

Girão, Luciana Facchinetti de Castro; Rocha, Surza Lucia Gonçalves da; Sobral, Ricardo Sposina; Bom, Ana Paula Dinis Ano; Sampaio, André Luiz Franco; Silva, José Godinho da; Ferrara, Maria Antonieta; Bon, Elba P. S.; Perales, Jonás

doi:10.1016/j.pep.2015.12.012

Cited by 21 publications

(9 citation statements)

References 36 publications

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“…A band around 45 kDa was detected in all samples collected after the induction (red arrow in Figure 6). This value is only a little higher than the molecular weight (38.7 kDa) reported for S. cerevisiae ASNase II, a tetramer formed by four identical subunits each containing 362 amino acid residues (Dunlop et al, 1978; Ferrara et al, 2010), likely due to protein glycosylation (Ferrara et al, 2006; de Castro Girão et al, 2016). In fact, despite the lack of the original L-asparaginase signal sequence, the bioproduct was expected to be secreted or to accumulate in the periplasm.…”

Section: Resultsmentioning

confidence: 70%

“…ASNase II activity was measured in whole cell suspension (periplasmic activity) based on asparagine hydroxylaminolysis, i.e., conversion of asparagine into β-aspartohydroxamate and ammonia. Samples (1.0 mL) of fermentation cultures were centrifuged at 3,320 g at 4°C for 10 min, and cells were resuspended in 0.9 mL of 20 mM Tris-HCl buffer (pH 6.8), 0.2 mL of 100 mM asparagine, and 0.2 mL of 1.0 M hydroxylamine (pH 7.0) (Dunlop et al, 1978; de Castro Girão et al, 2016). After incubation at 37°C for 30 min, 0.5 mL of trichloroacetic acid (TCA)/FeCl 3 reagent (50 g/L TCA and 100 g/L FeCl 3 in 0.66 M HCl) were added, and cell suspension centrifuged at 7,000 g for 10 min.…”

Section: Methodsmentioning

confidence: 99%

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Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUTs Strain

Rodrigues

Pillaca-Pullo

Torres-Obreque

et al. 2019

Front. Bioeng. Biotechnol.

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L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUTs under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L−1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.gglycerol-1 and a specific growth rate of 0.21 h−1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L−1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L−1, periplasmic specific enzyme activity 37.1 U.gdcw-1, volumetric enzyme activity 3,315 U.L−1, overall enzyme volumetric productivity 31 U.L−1.h−1, while the specific growth rate fell to 0.039 h−1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.

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Section: Resultsmentioning

confidence: 70%

Section: Methodsmentioning

confidence: 99%

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUTs Strain

Rodrigues

Pillaca-Pullo

Torres-Obreque

et al. 2019

Front. Bioeng. Biotechnol.

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“…2b) and is as expected for the protein backbone. l -Asparaginases from eukaryotic organisms such as fungi can also be glycosylated [12, 13]. However, it is reported that glycosylation patterns different to those in humans can trigger immune responses in patients [14].…”

Section: Discussionmentioning

confidence: 99%

Expression of a recombinant bacterial l-asparaginase in human cells

et al. 2019

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Objectivel-Asparaginase (ASNase) is an enzyme used in the treatment of acute lymphoblastic leukemia (ALL). As the therapeutic ASNases has bacterial origin, severe side effects are associated with its use, among them hypersensitivity and inactivation of the enzyme. In this context, the objective of this work was to produce a recombinant ASNase of bacterial origin in human cells in order to determine the presence and consequences of potential post-translational modifications on the enzyme.ResultsRecombinant ASNase was expressed in human cells with a molecular weight of 60 kDa, larger than in Escherichia coli, which is 35 kDa. N-glycosylation analysis demonstrated that the increased molecular weight resulted from the addition of glycans to the protein by mammalian cells. The glycosylated ASNase presented in vitro activity at physiological pH and temperature. Given that glycosylation can act to reduce antigenicity by masking protein epitopes, our data may contribute to the development of an alternative ASNase in the treatment of ALL in patients who demonstrate side effects to currently marketed enzymes.

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“…The wavelength 222 nm corresponds to the negative band signal typical of a-helix, and is a most-oen used wavelength to evaluate T m of a-helix dominated proteins. [38][39][40] Besides T m , the onset point (T onset ), where the thermal unfolding transition starts, was also investigated in this study. The second derivative of the CD data was obtained using Origin soware (Northampton, USA).…”

Section: Circular Dichroism (Cd) Analysismentioning

confidence: 99%

Conformational stability as a quality attribute for the cell therapy raw material human serum albumin

Wynendaele

Junren

et al. 2021

RSC Adv.

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Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris

Cited by 21 publications

References 36 publications

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUTs Strain

Fed-Batch Production of Saccharomyces cerevisiae L-Asparaginase II by Recombinant Pichia pastoris MUTs Strain

Expression of a recombinant bacterial l-asparaginase in human cells

Conformational stability as a quality attribute for the cell therapy raw material human serum albumin

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