Heterologous expression and purification of active L‐asparaginase I of <i>Saccharomyces cerevisiae</i> in <i>Escherichia coli</i> host

Santos, João H. P. M.; Costa, Iris Munhoz; Molino, João Vitor Dutra; Leite, Mariana Silva Moreira; Pimenta, Marcos de Mattos; Coutinho, João A. P.; Pessoa, Adalberto; Ventura, Sónia P. M.; Lopes, André Moreni; Monteiro, Gisele

doi:10.1002/btpr.2410

Cited by 14 publications

(9 citation statements)

References 38 publications

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“…Moreover, for recombinant His-tagged ASNases the immobilized metal ion affinity chromatography (IMAC), using a Ni2+-charged resin, is highly recommended. The use of IMAC proved to be an efficient tool in the purification of recombinant ASNase I of S. cerevisiae , resulting in high recoveries (40.50 ± 0.01%) and a purification factor of 17-fold (Santos et al, 2017).…”

Section: L-asparaginase Manufacturing: Production Process and Purificmentioning

confidence: 99%

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Brumano

Silva

Costa-Silva

et al. 2019

Front. Bioeng. Biotechnol.

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144

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L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

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Section: L-asparaginase Manufacturing: Production Process and Purificmentioning

confidence: 99%

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Brumano

Silva

Costa-Silva

et al. 2019

Front. Bioeng. Biotechnol.

Self Cite

144

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“…It is used as a fundamental drug in protocols for the treatment of acute lymphoblastic leukemia (ALL). Up to now, only bacterial enzymes either modified (PEGylated) from Escherichia coli or unmodified from E. coli and Erwinia chrysanthemi have been approved for ALL treatment (Ali et al, 2016; Lopes et al, 2017; Santos et al, 2017). Even with all the success achieved with bacterial ASNases, there are still adverse effects (Dunlop et al, 1978, 1980; Panosyan et al, 2004; Liu et al, 2016); therefore, there is a demand for new serologically different enzymes with improved characteristics such as less immunogenicity with similar or improved therapeutic effects (Narta et al, 2007).…”

Section: Introductionmentioning

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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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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“…IMAC has been frequently used as a capture step in l -ASNase purification processes. − This chromatography process was also used to purify recombinant proteins expressed in E. coli strain BL21 (DE3), the same strain used in our work.…”

Section: Resultsmentioning

confidence: 99%

Integrated Lab-Scale Process Combining Purification and PEGylation of l-Asparaginase from Zymomonas mobilis

Ramón

Gonçalves

Alvarenga

et al. 2021

Ind. Eng. Chem. Res.

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During the last quarter of a century, PEGylation has been used to enhance the clinical effectiveness of various therapeutic proteins. However, the conjugation process significantly increases the costs of the process. This parameter is critical, especially for biosimilar products that do not have patent protection. The integration of protein purification to its conjugation could potentially reduce total costs. Following this hypothesis, in this work, the purification of L-asparaginase from Zymomonas mobilis, produced recombinantly in Escherichia coli, linked to its conjugation with a PEG of 12 kDa, was studied. The effect of operating parameters on cell rupture by high-pressure homogenization was analyzed, and the optimal values for the conditions in this study were determined. The chromatographic capture and final purification steps were also studied. It was possible to elute the protein from ion-exchange chromatography, used as final purification, in the optimal buffer for the PEGylation step. This result allowed us to eliminate the buffer change step that is usually carried out at the beginning of the PEGylation process. The conjugate obtained by this continuous process showed higher thermal stability and slower protein degradation compared to the native protein. This continuous process not only has great potential to reduce costs but could also be useful for studying the effect of different parameters on the overall performance of the process. This knowledge is especially important to establish a quality by design approach at the industrial scale.

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Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

Cited by 14 publications

References 38 publications

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

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

Integrated Lab-Scale Process Combining Purification and PEGylation of l-Asparaginase from Zymomonas mobilis

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