Cloning, Expression And Characterization Of The α-Aspartyl Dipeptidase And Palmitoyl Protein Thioesterase1from Lampetra Japonica

Lampreys are one of the most ancient jawless vertebrates alive today whose habitat andbody structure have been highly conserved for at least350million years. In the present study,we report for the first time the molecular cloning and characterization of α-aspartyldipeptidase (AAD) and palmitoyl protein thioesterase1(PPT1) homolog from the Japaneselamprey (Lampetra japonica) leukocytes by the techniques of molecular biology andbioinformatics. They were analysis and predicted by molecular cloning, recombinantexpression and bioinformatics. Our researches may contribute to understand thecharacterization of the AAD and PPT1.AAD is able to hydrolyze N-terminal aspartal (Asp) dipeptides specificity. The twodipeptide sweeteners Aspartame and Alitame can be synthesis by the AAD. To date, theextensive studies that have been performed on the AAD in jawed vertebrates and bacterials,little is known about the biological activities of the AAD of L. japonica, one of the mostancient jawless vertebrates. In this study, we report for the first time the molecular cloningand characterization of the AAD homolog from the L. japonica. The DNA fragment ofLj-α-aspartyl dipeptidase (Lj-AAD) gene was cloned into pET-22b vector with a His-tag. Therecombinant Lj-AAD was expressed in Escherichia coli BL21(DE3) induced with1mMIPTG. The optimum conditions of the enzyme expression were optimized and therecombinant protein was identified by SDS-PAGE. It was showed that the ORF of Lj-AADwas717bp, and encodes a protein of238amino acids with an estimated molecular mass of26.5kDa in size and a theoretical isoelectrie point of5.83. The protein does’t contain a signalpeptide, existing in the form of inclusion bodies. The recombinant protein was successfullyrefolded and the activity was assayed. The pH dependency and thermal stability of theLj-AAD were investigated as the enzymatic properties. It was worth noting that Lj-AAD hadgood activity at low-temperature, can effectively improve the utilization of substract.PPT1is able to hydrolyze thioester linkages in palmitoyl-proteins, and removespalmitate residues by the lysosomal proteases degradation pathway. In present, only PPT1andacyl protein thioesterase1(APT1) are deemed to have function on depalmitoylation. In thisstudy, we report for the first time the molecular cloning and characterization of the PPT1homolog from the L. japonica. The ORF of Lj-PPT1gene was cloned into pET-22b vectorwith a His-tag. The recombinant Lj-PPT1was expressed in E. coli BL21(DE3) induced with1 mM IPTG. The optimum conditions of the enzyme expression were optimized and therecombinant protein was identified by SDS-PAGE. It was showed that the ORF of Lj-PPT1was972bp, and encodes a protein of323amino acids with an estimated molecular mass of36.6kDa in size and a theoretical isoelectrie point of5.91. The protein contains a signalpeptide, existing in both forms of inclusion bodies and supernatant.