Soluble Expression and Purification of Q59L Mutant L-asparaginase in the Presence of Chaperones in SHuffle™ T7 strain

Elham  Biglari Goliloo; Abdolnabi  Tollabi; Hossein  Zarei Jaliani

doi:10.18502/ijml.v8i2.6278

Elham Biglari Goliloo Department of Medical Biotechnology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Abdolnabi Tollabi Department of Medical Biotechnology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Hossein Zarei Jaliani Department of Medical Biotechnology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

DOI: https://doi.org/10.18502/ijml.v8i2.6278

Keywords: Asparaginase, Chaperone, E. coli, Shuffle

Abstract

Background and Aims: Q59L mutant of L-asparaginase enzyme from Escherichia coli (E. coli) has been introduced with lower side effects. This version of the enzyme might have potential applications in the treatment of leukemia patients. We utilized SHuffle T7 strain of E. coli, to produce the mutant enzyme in the presence of chaperone molecules.

Materials and Methods: Q59LAsp gene was cloned into pET28a expression vector, and two strains of E. coli (BL21 DE3 and SHuffle T7 strains) were used to produce recombinant protein. In parallel, PG-Tf2 plasmid was cloned into the same strains, and the effect of trigger factor chaperone and groELS chaperonines was studied. The his-tagged recombinant protein was expressed and purified using nickel affinity chromatography. The amount of recombinant protein which is produced in each condition was determined and compared.

Results: The amount of soluble recombinant protein was enhanced in the presence of chaperones in both strains of E. coli. SHuffle T7 strain produced more soluble recombinant protein in the soluble state than BL21 DE3 strain. So the best condition for the production of soluble recombinant Q59L mutant protein was the use of PG-Tf2 plasmid in the SHuffle T7 strain.

Conclusion: Application of the new strain SHuffle T7, with chaperones simultaneously showed better results in the production of Q59L mutant version of L-Asparaginase.