Cloning and expression of l-asparaginase from E. coli in eukaryotic expression system

Sajitha, Syed; Vidya, Jalaja; Karunakaran, Varsha; Pandey, Ashok

doi:10.1016/j.bej.2015.02.027

Cited by 40 publications

(27 citation statements)

References 16 publications

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“…Using genetic engineering, changes in residues not involved in the catalytic site, as the introduction of recombinant cysteine residues in the protein surface, are possible, which could, for example, improve ASNase PEGylation, and prevent dissociation-induced loss of activity (Ramirez-paz et al, 2018). Another strategy with great potential is the use of expression systems capable of expressing glycosylation patterns similar to those of mammals or even human-like (Sajitha et al, 2015; Nadeem et al, 2018). The glycosylation can improve the enzyme's pharmacokinetics, solubility, distribution, serum half-life, effector function, and binding to receptors, and can, therefore, be used to reduce many of the treatment side effects (Nadeem et al, 2018).…”

Section: Protein Engineering For Improvement Of L-asparaginase Therapmentioning

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.

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: Protein Engineering For Improvement Of L-asparaginase Therapmentioning

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.

144

View full text Add to dashboard Cite

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“…On the other hand, the enzyme produced by C. utilis has k M value of 77 μM 78 . In a recent study, Soler et al (2015) 7 tested 43 different strains of yeasts, verifying that only strains of Issatchenkia orientalis and Rhodotorula glutinis showed periplasmic l -asparaginase activity when growth in liquid CD-m. Also, Sajitha et al (2015) 79 presented an investigation by using an expression study of gene ansB of E. coli , which encodes l -asparaginase enzyme, in yeast. This study was developed on a new protein expression system based on the yeast Pichia pastoris .…”

Section: L-asparaginase Applicationsmentioning

confidence: 99%

Current applications and different approaches for microbial l-asparaginase production

Cachumba

Antunes

Peres

et al. 2016

Brazilian Journal of Microbiology

174

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l-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in l-aspartic acid and ammonium. This enzyme is presented in different organisms, such as microorganisms, vegetal, and some animals, including certain rodent's serum, but not unveiled in humans. It can be used as important chemotherapeutic agent for the treatment of a variety of lymphoproliferative disorders and lymphomas (particularly acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma), and has been a pivotal agent in chemotherapy protocols from around 30 years. Also, other important application is in food industry, by using the properties of this enzyme to reduce acrylamide levels in commercial fried foods, maintaining their characteristics (color, flavor, texture, security, etc.) Actually, l-asparaginase catalyzes the hydrolysis of l-asparagine, not allowing the reaction of reducing sugars with this aminoacid for the generation of acrylamide. Currently, production of l-asparaginase is mainly based in biotechnological production by using some bacteria. However, industrial production also needs research work aiming to obtain better production yields, as well as novel process by applying different microorganisms to increase the range of applications of the produced enzyme. Within this context, this mini-review presents l-asparaginase applications, production by different microorganisms and some limitations, current investigations, as well as some challenges to be achieved for profitable industrial production.

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“…The proper protein folding, activity, purity and yield are dependent on the host ability to translate, fold and keep high amounts of the desirable protein in its soluble form. Sajitha et al 24 successfully expressed the ASNase from E. coli into the eukaryotic host P. pastoris. The authors obtained a soluble and active ASNase in the supernatant and speculate that this protein might undergo eukaryotic glycosylation conferring more stability to the protein and fewer side effects.…”

Section: Expression and Purification Of Sc_asnaseimentioning

confidence: 99%

“…The hypersensitivity reactions ranging from a small allergy in the drug injection area, bronchospasm and even anaphylactic shock, occurs in 5–50% of the treated patients. Nowadays, the researchers are focused in the partial or total elimination of these problems, regarding some protein structural modification approaches, through genetic engineering, gene enhancement and/or by ASNase conjugation with biopolymers ( e.g., polyethylene glycol (PEG), dextran, albumin and heparin) or by changing the expression host systems …”

Section: Introductionmentioning

confidence: 99%

“…Nowadays, the researchers are focused in the partial or total elimination of these problems, regarding some protein structural modification approaches, through genetic engineering, gene enhancement and/or by ASNase conjugation with biopolymers (e.g., polyethylene glycol (PEG), dextran, albumin and heparin) 5,11,[21][22][23] or by changing the expression host systems. 24 In this work, the genes codifying ASNase were isolated from the Saccharomyces cerevisiae BY4741 (Invitrogen), a microorganism able to synthesize two distinct forms of ASNase: Sc_ASNaseI, an intracellular constitutive enzyme and Sc_ASNaseII, a periplasmic enzyme which is secreted when the microorganism is exposed to some stress conditions, in particular to nitrogen starvation. Both enzymes are poorly characterized; Ferrara et al 25 obtained a better endogenous amount of Sc_ASNaseII using the double mutant strain Dure2Ddal80.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Santos

Costa

Molino

et al. 2016

Biotechnology Progress

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l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His) -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017.

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Cloning and expression of l-asparaginase from E. coli in eukaryotic expression system

Cited by 40 publications

References 16 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

Current applications and different approaches for microbial l-asparaginase production

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

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