A Protein Disulfide Isomerase from Bacillus Subtilis DR8806: Gene Cloning, Expression,Purification and Biophysicochemical Characterization

The correct folding of proteins is one of the most important problems in the production of recombinant pro-teins. The existence of disulfide bonds in the proteins structure is the important factor leading to improper folding of proteins while formation of correct disulfide bonds is required for the biological activity of many proteins Protein disulfide isomerase is the first and most important folding catalyst of proteins and catalyze reduction, formation and isomerization of disulfide bonds. In this study, the gene encoding protein disulfide isomerase was cloned as fusion with glutathione S transferase into pGEX4t vector and expressed in Escherichia coli TOP10. The gene was from strain Bacillus subtilis DR8806 which has been isolated from hot mineral spring in Kerman. The enzyme was purified by GST affinity chromatography with a specific activity of 1227 U/mg. The enzyme se-quence showed an open reading frame of 618 bp encoding a 206 amino acid, and about 5%, it is structurally dif-ferent from all the proteins which have been reported. The molecular weight of the purified enzyme was 23.26 kDa. The enzyme activity increased in the presence of Mn+2 and Co+2 ions while Ag+ and Zn +2 ions decreased not have any effect on its activity. Some of the known inhibitors of protein the activity, Ba+2 and Ca+2 ions did while others did not have such effects . The enzyme was active disulfide isomerase inhibited enzyme activity and stable at the wide range of pH (4.5-10) and temperature (20-85◦C) with the optimum pH 5.5 and optimum temperature 65◦C. No reports have been presented regarding the stable characteristics of the protein disulfide capabilities for the therefore this enzyme has potential ,isomerase with a wide range of pH and temperature .respective industries

کلیدواژه ها:

Bacillus subtilis ، Cloning ، Expression ، Protein Disulfide Isomerase