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Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

Abstract：

:Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Acero-Navarro KE,Jiménez-Ramírez M,Villalobos MA,Vargas-Martínez R,Perales-Vela HV,Velasco-García R

doi

10.1016/j.pep.2017.10.004

subject

Has Abstract

pub_date

2018-02-01 00:00:00

pages

53-61

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(17)30020-7

journal_volume

142

pub_type

杂志文章

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