Molecular Cloning, Expression And Analysis Of A Prolyl Endopeptidase From Haliotis Discus Hannai

Prolyl endopeptidase?PEP/PREP?belongs to the serine protease family and is of great potential application value in medicine and food science.In this study,the full-length cDNA sequence of prolyl endopeptidase?Hdh-PEP?was cloned from abalone Haliotis discus hannai.Hdh-PEP was expressed in vitro,and the properties of expressed Hdh-PEP were studied.Specific primers were designed based on the analysis of conserved sequences in PEP genes of multiple species.The full-length cDNA sequence of prolyl endopeptidase from the hepatopancreas of Haliotis discus hannai was cloned using polymerase chain reaction?PCR?and rapid amplification of cDNA ending technologies.Sequence analysis showed that the cDNA of Hdh-PEP was 3224 base pairs?bp?in length,including 5'-UTR of 51 bp,3'-UTR of 1052 bp and an ORF of 2121 bp,encoding 706 amino acid residues.The sequence alignment showed the similarities of amino acid sequence of Hdh-PEP and PEP in Crassostrea gigas,Xenopus tropicalis and Homo sapiens were 70%,65%and 63%,respectively.According to the prediction,the molecular weight of Hdh-PEP was 80.3 ku and its theoretical isoelectric point was 5.55 as a relatively stable hydrophilic protein.The secondary and tertiary structure simulations of Hdh-PEP showed that it had typical PEP family characteristics,a"cassette-like"structure contained an"?-catalytic domain"and a"?-propeller domain".Phylogenetic analysis showed that Hdh-PEP formed an independent evolutionary branch in gastropod PEP.A prokaryotic recombinant vector pET-28a-HP was constructed by ligating the full length sequence encoding Hdh-PEP to the pET-28a vector,for the in vitro expression.Recombinant Hdh-PEP with enzyme activity was expressed and purified.The recombinant Hdh-PEP with activity was expressed and purified.The theoretical relative molecular weight of the recombinant Hdh-PEP was 85.4 ku including the weight of original Hdh-PEP?80.3 ku?and the weight of His-tag and vector promoters?4.1 ku?,which was consistent with the result on SDS-PAGE.The results of peptide mass fingerprinting mass spectrometry analysis were 100%identified to the amino acid of theoretical sequence in the molecular cloning results,which showed that the expression product was correct.Enzymatic properties analysis showed that the optimal temperature and optimum pH of Hdh-PEP were 20°C and 6.0,respectively,and the relative high enzyme activity was maintained in the low temperature range?10°C-20°C?and pH 6.0-9.0,and rapidly deactivated at temperatures above 30°C.Hdh-PEP specifically cleaved the fluorescent substrate Suc-Gly-Pro-MCA and Suc-Gly-Pro-Leu-Gly-Pro-MCA containing proline residues at the carboxyl terminus.The specific activity of Hdh-PEP was 6.57 U/?g under optimum conditions.Inhibitors and metal ions were used to analyze the inhibitory effect on Hdh-PEP.The specific inhibitor SUAM-14746 strongly inhibited the activity of Hdh-PEP,and Na⁺and Mg²⁺did not have a significant effect on the activity of Hdh-PEP while Ca²⁺promoted a little.The inhibitory effects of EGAR and silibinin on Hdh-PEP were relatively weak.SUAM-14746showed competitive inhibition to Hdh-PEP and Cu²⁺revealed mixed inhibition to Hdh-PEP.Zn²⁺and Al³⁺had an effect on the secondary structure when Hdh-PEP activity was inhibited,while Cu²⁺,Pb²⁺and Ag⁺did not significantly affect the secondary structure when Hdh-PEP activity was inhibited.The expression level of Hdh-PEP in different tissues of Haliotis discus hannai was detected by Western Blot.The results showed that the rabbit-anti-PEP?Oreochromis spp?polyclonal antibody had specific immuno-hybridization responses to both native and recombinant Hdh-PEP.The expression level of Hdh-PEP in blood cells was relatively high and was less in gill tissue.However,Hdh-PEP expression was not detected in gastropod muscle.The PEP storage solution formulation from Sigma-Aldrich was modified to prepare storage buffers.The results show that the modified storage buffer can maintain Hdh-PEP activity above90%within 75 days.In this study,molecular cloning of PEP from Haliotis discus hannai supplemented the research gaps of shellfish PEP and we established a stable expression system for the further researches,which may provide a certain theoretical basis for the follow-up study of shellfish PEP.