Change of the N-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human D-amino acid oxidase as active inclusion bodies.

Abstract

To optimize the production of active inclusion bodies (IBs) containing human D-amino acid oxidase (hDAAO) in Escherichia coli. The optimized initial codon region combined with the coexpressed rare tRNAs, fusion of each of the N-terminal partners including cellulose-binding module, thioredoxin, glutathione S-transferase and expressivity tag, deletion of the incorporated linker, and improvement of tRNA abundance affected the production and activity for oxidizing D-alanine of the hDAAO in IBs. Compared with the optimized fusion constructs and expression host, IBs yields and activity were increased to 2.6- and 2.8-fold respectively by changing the N-terminal codon bias of the hDAAO. The insoluble hDAAO codon variant displayed the same substrate specificity as the soluble one for oxidizing D-alanine, D-serine and D-aspartic acid. The freshly prepared hDAAO codon variant was used for analyzing the L-serine racemization activity of the bacterially expressed maize serine racemase. Optimization of the N-terminal codon bias combined with the coexpression of rare tRNAs is a novel and efficient approach to produce active IBs of the hDAAO.