Cloning, purification and biochemical characterization of two glutathione S-transferase isoforms from Rutilus frisii kutum

Salman Ahmadi; Hossein Ghafouri; Sara Tarazi; Sajjad Sarikhan; Omid Saberi Kh

doi:10.1016/j.pep.2020.105800

Cloning, purification and biochemical characterization of two glutathione S-transferase isoforms from Rutilus frisii kutum

Protein Expr Purif. 2021 Mar:179:105800. doi: 10.1016/j.pep.2020.105800. Epub 2020 Nov 25.

Authors

Salman Ahmadi¹, Hossein Ghafouri², Sara Tarazi¹, Sajjad Sarikhan³, Omid Saberi Kh¹

Affiliations

¹ Department of Biology, Faculty of Basic Sciences, University of Guilan, Rasht, Iran.
² Department of Biology, Faculty of Basic Sciences, University of Guilan, Rasht, Iran; Department of Marine Sciences, The Caspian Sea Basin Research Center, University of Guilan, Rasht, Iran. Electronic address: h.ghafoori@guilan.ac.ir.
³ Molecular Bank, Iranian Biological Resource Center (IBRC), ACECR, Tehran, Iran.

PMID: 33248224
DOI: 10.1016/j.pep.2020.105800

Abstract

Glutathione S-transferases are an important multifunctional family of intracellular enzymes that their detoxification function has been reported in fishes since 1970, but no studies have been conducted on Rutilus frisii kutum GSTs yet. In the present study, RkGSTA and RkGSTM encoding genes were cloned and sequenced and their nucleotide sequences were submitted to NCBI GenBank. In order to reduce the expression challenges of recombinant proteins including low solubility, low yield and insufficient purity issues in E. coli, the pKJE7 chaperone plasmid was used to increase the recovery of expressed proteins in the soluble fractions. Best expression clone was selected for purification by Ni-NTA affinity chromatography. The three-dimensional structural models were constructed by I-TASSER. The optimum temperature of purified RkGSTA and RkGSTM was 35 and 30 °C, with optimum activity at pH 9.0 and 8.5, respectively. The thermostability and pH stability results indicated that RkGSTA is more heat-tolerant than RkGSTM though both of them retained more than 80% of their activities at pH 6.5 to 9.0. Overall, this study represents a comprehensive perspective on the structural and biochemical aspects of this enzyme that would be even used in further researches such as drug design studies in order to eliminate toxicant compounds from the body and environment of fishes to protect them against undesired harmful damages.

Keywords: Caspian white fish; Detoxification; Glutathione S-Transferase; Protein expression; Protein purification; Rutilus frisii kutum.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Chromatography, Affinity
Cypriniformes / genetics*
Enzyme Stability
Fish Proteins* / chemistry
Fish Proteins* / genetics
Fish Proteins* / isolation & purification
Fish Proteins* / metabolism
Glutathione Transferase* / chemistry
Glutathione Transferase* / genetics
Glutathione Transferase* / isolation & purification
Glutathione Transferase* / metabolism
Hydrogen-Ion Concentration
Models, Molecular
Protein Isoforms
Recombinant Proteins* / chemistry
Recombinant Proteins* / genetics
Recombinant Proteins* / isolation & purification
Recombinant Proteins* / metabolism
Temperature

Substances

Fish Proteins
Protein Isoforms
Recombinant Proteins
Glutathione Transferase