Dataset Information

Expression of a recombinant bacterial L-asparaginase in human cells.

ABSTRACT: OBJECTIVE:L-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. RESULTS:Recombinant 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.

SUBMITTER: Dantas RC

PROVIDER: S-EPMC6896745 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Expression of a recombinant bacterial L-asparaginase in human cells.

Dantas Raquel Caminha RC Caetano Ludmilla Freire LF Torres Ariany Lima Sousa ALS Alves Matheus Soares MS Silva Emanuelly Thays Muniz Figueiredo ETMF Teixeira Louhanna Pinheiro Rodrigues LPR Teixeira Daniel Câmara DC de Azevedo Moreira Renato R Fonseca Marcela Helena Gambim MHG Gaudêncio Neto Saul S Martins Leonardo Tondello LT Furtado Gilvan Pessoa GP Tavares Kaio Cesar Simiano KCS

BMC research notes 20191205 1

<h4>Objective</h4>L-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 ...[more]

PMID: 31806048

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Project description:BackgroundTransmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms.ResultsPlasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices.ConclusionsIn this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this one could aid in overcoming the technical bottlenecks in working with TM proteins and could potentially aid in increasing the rate of structure determination.

Dataset Information

Expression of a recombinant bacterial L-asparaginase in human cells.

Publications

Expression of a recombinant bacterial L-asparaginase in human cells.

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