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. 2015 Dec 12;8(12):e17790.
doi: 10.5812/jjm.17790. eCollection 2015 Dec.

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

Ali Mohammad Latifi  1 , Khosro Khajeh  2 , Gholamreza Farnoosh  1 , Kazem Hassanpour  3 , Samaneh Khodi  1
Affiliations

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

Ali Mohammad Latifi et al. Jundishapur J Microbiol. .

Abstract

Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process.

Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b).

Materials and methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd.

Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band.

Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step.

Keywords: Cytoplasmic; Escherichia coli; Organophosphorus; Periplasmic.

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Figures

Figure 1.
Figure 1.. Double-Digestion of Recombinant Plasmids
Lane 1, standard molecular weight marker; lane 2, pET21a-opd double digestion; lane 3, pET26b-opd double digestion.
Figure 2.
Figure 2.. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Analysis of pET21a-opd and pET32a-opd Soluble and Insoluble Fractions
The fractions were expressed in E. coli Rosetta-gami cells with 0.5 mM IPTG for seven hours at 18°C, 30°C and 37°C. M, molecular weight marker; P, pellet; S, soluble.
Figure 3.
Figure 3.. The Activity Assay of Soluble Fractions of pET21a-opd and pET26b-opd Enzymes
Compressing the activity at three temperatures, the highest activity was observed at 30°C and the lowest at 18°C.
See this image and copyright information in PMC

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