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Prolidase is a metallopeptidase that is ubiquitous in nature and has been isolated from mammals, bacteria and archaea. Prolidase specifically hydrolyzes dipeptides with a prolyl residue in the carboxy terminus (NH(2)-X-/-Pro-COOH). Currently, the only solved structure of prolidase is from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme is of particular interest because it can be used in many biotechnological applications. Prolidase is able to degrade toxic organophosphorus (OP) compounds, namely, by cleaving the P-F and P-O bonds in the nerve agents, sarin and soman. Applications using prolidase to detoxify OP nerve agents include its incorporation into fire-fighting foams and as biosensors for OP compound detection. Prolidases are also employed in the cheese-ripening process to improve cheese taste and texture. In humans, prolidase deficiency (PD) is a rare autosomal recessive disorder that affects the connective tissue. Symptoms of PD include skin lesions, mental retardation and recurrent respiratory infections. Enzyme replacement therapies are currently being studied in an effort to optimize enzyme delivery and stability for this application. Previously, prolidase has been linked to collagen metabolism and more recently is being associated with melanoma. Increased prolidase activity in melanoma cell lines has lead investigators to create cancer prodrugs targeting this enzyme. Thus, there are many biotechnological applications using recombinant and native forms of prolidase and this review will describe the biochemical and structural properties of prolidases as well as discuss their most current applications.

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.

Complete PCR-derived DNA fragments containing the structural genes for DNA polymerases of the archaeons Pyrococcus furiosus and Pyrococcus woesei were cloned into an expression vector. The clones expressing thermostable His-tagged DNA polymerases were selected. The cloned fragments were sequenced. The DNA sequences were verified to be authentic by sequencing several clones. The nucleotide (nt) sequence revealed that DNA polymerase of P. woesei (Pwo DNA polymerase) consists of 775 amino acids and has a molecular weight of 90,566. It shows 100% nucleotide identity to the nucleotide sequence of DNA polymerase from P. furiosus (Pfu DNA polymerase). The results confirm that nucleotide sequences of both archaeons (P. furiosus and P. woesei) are highly similar. The recombinant DNA polymerases (His-tagged Pfu and His-tagged Pwo) contained a polyhistidine tag at the N-terminus (43 additional amino acids) that allowed single-step isolation by Ni-affinity chromatography. We found that recombinant plasmids are toxic or unstable in the expressing strain BL21(DE3), even in the absence of the inducing agent, IPTG. However, the plasmids were stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were expressed upon IPTG addition. The proteins were purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni2+-Sepharose columns. The enzymes were characterized and displayed high DNA polymerase activity and thermostability. This bacterial expression system appears to be the method of choice for production of Pfu or Pwo DNA polymerases., (Copyright 1998 Academic Press.)

The monoclinic crystal form of methionine amino-peptidase from Pyrococcus furiosus (MAP-Pfu) has been crystallized from four different conditions. Native crystals belong to space group P2(1) with typical unit-cell dimensions a = 53.4, b = 85.1, c = 72.7 A, beta = 107.7 degrees and diffract to 2.9-4.5 A resolution. However, there is a problem of nonisomorphism among the crystals. Water-mediated transformation to low-humidity form occurs by reduction of the relative humidity of crystal environment to 79%. The unit-cell dimensions of transformed crystals are a = 51.9, b = 83.3, c = 70.3 A, beta = 105.9 degrees, and the calculated solvent content is 3.9% less than in original crystals. Transformation to low-humidity form is accompanied by 1.7 times reduction of overall temperature factors, extension of diffraction resolution up to 1.75 A, without change or reduction of crystal mosaicity, and improvement in stability to X-ray radiation. The water-mediated transformation also appears to relieve the problem of nonisomorphism among the original MAP-Pfu crystals.

Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) amplification. Previously the protein has been purified from P. furiosus bacterial cultures, and a recombinant form has been produced in a baculovirus system. We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC, Accession No. 87496) and found that this plasmid is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction. However, the plasmid was stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90-kDa protein was expressed upon addition of isopropyl beta-D-thiogalactopyranoside. The protein was purified by heating (to denature E. coli proteins), followed by chromatography on P11 phosphocellulose and mono Q columns. The purified protein had the same activity as the commercially obtained baculovirus-expressed Pfu in both DNA polymerase and PCR reactions. This bacterial expression system appears to be the method of choice for production of Pfu., (Copyright 1997 Academic Press.)