Optimized codons of polar tube protein 1 gene of Encephalitozoon cuniculi to enhance protein expression in Escherichia coli

Abstract

Encephalitozoon cuniculi (E. cuniculi) is an intracellular zoonotic parasite that is recognized as a common parasite of rabbits. Infected rabbits show neurological and renal disorder. The polar tube protein 1 of the spore is revealed to be antigenicity. The present study aimed to produce a recombinant polar tube protein 1 (rPTP1) for which the codon usage of a native ptp1 gene was optimized and synthesized for protein production in Escherichia coli (E. coli). Both a native ptp1 gene amplified by PCR technique and an optimized ptp1 gene were ligated into a pQE30 expression vector and transformed into an E. coli strain M15. The rPTP1 production was induced by 1M isopropyl--D-thiogalactoside. A protein band with a molecular weight of about 45 kDa was found in the extracted protein of both E. coli transformed with optimized ptp1 and native ptp1 plasmids. SDS-PAGE and western blot analysis using an anti-histidine antibody revealed a notably higher protein yield of the E. coli containing an optimized ptp1 plasmid than that of the E. coli containing a native ptp1 plasmid. Biological function of the recombinant protein was evaluated by western blotting using E. cuniculi-positive and negative rabbit sera. Result showed that the biological function of the optimized rPTP1 protein expressed from E. coli could differentiate between E. cuniculi-negative and positive rabbit sera. Therefore, rPTP1 can be used for the development of a diagnostic test for E. cuniculi infection in the future.