High Level Production Of Human DAAO As Active Inclusion Bodies For Coupled Assay Of Maize Serine Racemase

D-Amino acid oxidase(DAAO),containing non-covalently bound FAD as the cofactor,catalyzes the oxidative deamination of D-amino acids to the corresponding keto acids,NH4+ and H2O2.Owing to their broad substrate specificity,eukaryotic DAAOs have various applications in the field of industry,biosynthesis,analysis and medicine.Expression of active inclusion bodies(IBs)is a novel method for producing carrier-free protein immobilizate.This method have several advantages such as high production,enhanced protein stability,easier purification.In this experiment,we tried to construct different human DAAO(hDAAO)genes to produce active IBs containing hDAAO in Escherichia coli,detect and compare the differences in activity and production.The results are as follows:1.Construction of the hDAAO protein: The hDAAO and codon mutant genes were cloned and synthesized,four N-terminal chaperones including cellulose-binding module,thioredoxin,glutathione S-transferase and oligopeptide containing N-terminal seven amino acid residues(MTDVTIK)of E.coli translation initiation factor II were integrated upstream in the expression of wild hDAAO.Tobacco etch virus protease(TEVp)recognition sequence tevS(ENLYFQG)was added between the tags and the target protein.2.Overexpression and activity analysis of recombinant protein: The constructed recombinant plasmids containing the target protein were expressed in E.coli BL21(DE3)and Rosetta(DE3),respectively.The effects of fusion of the N-terminal partners,deletion of the incorporated linker,improvement of tRNA abundance and changes of codon bias on the yields and activity were analyzed.The results showed that all added tags or sequences have different impacts on the production and activity of the hDAAO in IBs.Change of the N-terminal codon bias combined with tRNA supplementation outperformed the selected fusion tags for production of hDAAO as active inclusion bodies.3.Substrate specificity detection: Differences in activity of the selected three D-amino acids(D-alanine,D-serine and D-aspartate)catalyzed by hDAAO active mutant showed that insoluble aggregates had no significant effect on substrate specificity changes.4.Coupled assay of the maize serine racemase activity: The purified hDAAO codon mutant protein were mixed with the purified recombinant Zea mays serine racemase(ZmSR),the L-serine was used as substrate.The products catalyzed by hDAAO were determined.The results suggested that purified hDAAO active inclusion bodies was more efficient than the DAAO commercially extracted from porcine kidney.In summary,we found a novel and effective approach for the production of hDAAO active inclusion bodies using optimization of the N-terminal codon bias combined with coexpression of rare tRNAs.It is promising to be used for converting proteins or enzymes that are less efficiently folded in E.coli expression into active IBs.