Soluble expression and purification of a full-length asparaginyl tRNA synthetase from Fasciola gigantica.

ABSTRACT

We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including humans. Aminoacyl tRNA synthetases (AARS) catalyze the first step of protein synthesis. They attach an amino acid to its cognate tRNA, forming an amino acid-tRNA complex. The gene that codes for FgNRS was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pQE30 under the transcriptional control of the bacteriophage T5 promoter and lac operator. M15 Escherichia coli strain transformed with the FgNRS expression vector pQE30-NRS accumulates large amounts of a soluble protein of about 61 kDa. The protein was purified to homogeneity using immobilized metal affinity chromatography. The recombinant protein was further confirmed by immunoblotting with anti-His antibody. Following size exclusion chromatography, the FgNRS was stable and observed to be a dimeric protein. In this study, the expression and purification procedures have provided a simple and efficient method to obtain full-length FgNRS in large quantities. This will provide an opportunity to study the structure, dynamics and function of NRS.