Cloning And Expression Of Gene Encoding Human Liver Prolidase And Biochemico-pharmacological Properties Of Rh-Prolidase

The specificity and extreme toxicity of organophosphorus (OP) compounds such as soman, sarin, tabun, parathion and paraoxon have led to their development as chemical warfare agents as well as agricultural pesticides. Current antidotal treatments against the poisoning include pretreatment with reversible AChE inhibitors, post-exposure therapy with anticholinergic drugs to counteract the effects of excess acetylcholine and treatment with oximes to reactivate OP-inhibited AchE. Although these antidotal treatments are effective in preventing lethality from OP exposure, they do not effectively prevent the post-exposure incapacitation and toxic effects that are commonly observed in experimental animals and are likely to occur in humans. One new approach is to rapidly sequester OP compounds with enzymes and thereby detoxify them in the blood before they can reach their physiological targets, acetylcholinesterase and cholinorecepter in the nervous system. Organophophorus acid anhydrolase (OPAA) have been found in a wide variety of prokaryotic and eukaryotic organisms, and especially, with abundant activities in animal liver and kidney. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of very toxic chemical nerve agents.A sort of OPAA from human liver was purified by Dr. Wang Qing-ding in our lab. The N-terminal amino acid sequence of 10 residues was determined by SDS-PAGE, electroblotting, and subsequent sequence analysis. Amino acid sequence comparison with the primate database showed that the N-terminal amino acid sequence of OPAA is similar to the N-terminal amino acid sequence of human liver prolidase from amino acid residue 9 to 18 with a similarity of 90%, except W→P change at the residue 2 of the OPAA. On the other hand, Zheng Tu-chen (US Army Chemical and Biological Defense Agency) reported that a moderately halophilic bacterial isolate Alteromonas JD6.5 was found to possess high levels of OPAA activity against several highly toxic organophosphorus compounds including soman and sarin. Amino acid sequence comparison showed significant structural similarity between the OPAA and prolidase. The structural homology and functional similarities suggest that the OPAA and prolidase may have evolved from the same ancestral gene.In this study, human liver prolidase gene was cloned from the liver of a healthy adult. Three kinds of human prolidase expression plasmids were constructed using DNA recombinant technique to express recombinant human prolidase(rh-prolidase) inE. coli, S. cerevisiae and P. pastoris, respectively, rh-prolidase was purified by ion exchange gradient chromatography and gel filtration chromatography. The biochemical pharmacological characteristics of rh-prolidase were studied.1 Two pairs of primers were designed according to the human prolidase nucleotide sequence from literature(Endo F, 1989) for cloning of the prolidase gene. Total RNA was purified from liver, and the prolidase cDNA was amplified by RT-PCR with the primers using the RNA as template. The prolidase gene was inserted into the PinPoint-T, and the full-length sequence was verified.2 Prolidase gene was then amplified by PCR from the recombinant plasmid PinPoint-P, and was cloned into pGEX4T-1 vector to construct pGEX4T-1-P expression plasmid. pGEX4T-1-P was transformed into competent cells of E. coli BL21, and recombinant strains were induced by IPTG to express GST fusion prolidase. SDS-PAGE analysis revealed that the rh-prolidase was highly expressed and accumulated up to 21% of the total amount of bacterial proteins. The rh-prolidase existed in the form of inclusion body. The inclusion bodies were isolated, denatured and refolded, however, the treated protein did not show any enzyme activity.3 To assess the enzyme catalytic characteristics of the expressed products, human liver prolidase gene was inserted into S. cerevisiae expression vector pYES2 to construct the recombination expression vector pYES2-P, and then it was transformed into S. cerevisiae INVScl by electroporation. Transformant w-3 with the highest enzymatic activity was induced by galactose for expression. The optimal induction conditions such as temperature, induction time and initial OD600 were determined by orthogonal experimental design L9(34). SDS-PAGE of the disrupted cell supernatants showed that prolidase gene expressed a protein of 56 kDa. Intensity scanning of the SDS-PAGE gel revealed that the targeted protein accounted for 3.16% of the total protein in the supernatant. One liter induction medium could produce 7g of wet yeast cell in which there was 4.56mg of recombination protein. The recombinant enzyme was assayed for the prolidase and OPAA activities. The results showed that the recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions, the maximal activities of prolidase and OPAA were 226.5 and 578 nmol/min/mg in the cell lysate supernatants, respectively.4 The human liver prolidase gene was also cloned into the pPIC9K, expression vector of P. pastoris. The recombinant expression vector pPIC9K-P was at first linearized by restriction enzyme Bg1 II and Pme I, respectively, and then transformedinto P pastoris GS115 by electroporation. Two kinds of transformants (the transplacement recombinant and the insertion recombinant) were subjected to screening. Both recombinants were induced for expression by methanol. The insertion recombinant P2# induction conditions such as medium pH, methanol concentration and induction time were determined by orthogonal experimental design L9(34). SDS-PAGE analysis showed that prolidase gene expressed a protein of 73 kDa. Intensity scanning of the SDS-PAGE gel revealed that the targeted protein accounted for 55% of the total protein in the supernatant. One liter induction medium could produce 86.2 mg of recombination protein. The results of activity assay confirmed that the recombinant enzyme could catalyze soman and Gly-Pro. The maximal activities of prolidase and OPAA came to 44.1 and 54.8 nmol/min/mg in the medium supernatant, respectively.5 rh-prolidases from the P2# and w-3 supernatants were separately purified by ion exchange gradient chromatography (DEAE-Sepharose Fast Flow) and gel filtration chromatography (Sephacryl S-200 High Resolution). The purified recombinant protein showed a single band by SDS-PAGE analysis. One soman lethal dose of Kunming mice was tested to be 200 μg/kg. When soman (final concentration 0.19 mM) was preincubated with rh-prolidase in vitro at 37℃ for 15 min and then injected subcutaneously to mice at a dose of 1.0 LD, all the mice survived, whereas the control mice died within 10 min. The stabilities of rh-prolidase activity at various temperatures were also investigated.