The Efficient Solubilization and Refolding of Recombinant Organophosphorus Hydrolyses Inclusion Bodies Produced in Escherichia coli

Mirhosseini, Seyed Ali; Latifi, Ali Mohammad; Mahmoodzadeh Hosseini, Hamideh; Seidmoradi, Rezvan; Aghamollaei, Hossein; Farnoosh, Gholamreza

doi:10.29252/JABR.06.01.04

The Efficient Solubilization and Refolding of Recombinant Organophosphorus Hydrolyses Inclusion Bodies Produced in Escherichia coli

Document Type : Original Article

Authors

Seyed Ali Mirhosseini ¹

Ali Mohammad Latifi ²

Hamideh Mahmoodzadeh Hosseini ¹

Rezvan Seidmoradi ³

Hossein Aghamollaei ⁴

Gholamreza Farnoosh ²

¹ Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

² Applied Biotechnology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

³ Faculty of Biological Science, Alzahra University, Tehran, Iran

⁴ Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

10.29252/JABR.06.01.04

Abstract

Introduction: Organophosphorus hydrolase(OPH) is an enzyme that can degrade organophosphorus compounds in pesticides. High expression levels of OPH in Escherichia coli lead to form inclusion body in cytoplasmic space which is an inactive form of protein and needs a solubilizing and refolding process. The aim of this study was to compare different methods for solubilization and refolding of recombinant OPH expressed in E. coli.
Materials and Methods: OPH was expressed in E. coli and purified by the Ni-NTA column. The refolding efficiency of this protein was assessed by 4 strategies: dialysis, rapid dilution, on column and combination of rapid dilution and dialysis. In each case, the refolding efficiency was evaluated by SDS-PAGE analysis and enzyme activity assay and was compared to find the best procedure.
Results: The refolding efficiency of these 4 strategies was estimated at about 12%, 10%, 14% and 50% for on column, rapid dilution, dialysis and combination of rapid dilution and dialysis, respectively. Results showed that during the refolding process, most proteins did not reach the correct structure and aggregated again while the combination of 2 methods, rapid dilution and dialysis provided an appropriate procedure to refold.
Conclusions: The combination of rapid dilution and dialysis is an efficient method for refolding OPH. The efficacy of this method for refolding other recombinant proteins can be further investigated.

Keywords

Organophosphorus Hydrolases

Inclusion Bodies

Solubilization

Refolding

Protein Activity