Abstract
Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described.
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References
Andrews, PC, Hines CM, Dixon JE (1980) Characterization of proline endopeptidase from rat brain. Biochemistry 257:5861–5865
Blumenberg S, Teichberg VI, Charli JL, Hersh LB, McKelvy JF (1980) Cleavage of substance P to an N-terminal tetrapeptide and a C-terminal heptapeptide by post-proline cleaving enzyme from bovine brain. Brain Res 192:477–486
Boer H de, Comstock LJ, Vasser N (1983) The tac promoter: functional hybrid derived from the trp and lac promoters. Biochemistry 80:21–25
Bradford M (1976) A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Breddam K (1986) Serine carboxypeptidase. A review. Carlsberg Res Commun 51:83–128
Breddam K, Sørensen SB, Svendsen I (1987) Primary structure and enzymatic properties of carboxypeptidase II from wheat bran. Carlsberg Res Commun 52:297–311
Brenner S (1988) The molecular evolution of genes and proteins: a tale of two serines. Nature 334:528–530
Bullock WO, Fernandez JM, Short JM (1987) XL 1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5:376–379
Chevallier S, Goeltz P, Thibault P, Banville D, Gagnon J (1992) Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. J Biol Chem 267:8192–8199
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 133:6–13
Gottesman S, Halpern E, Trisler P (1981) Role of sulA and SulB in filamentation by lon mutations of Escherichia coli K12. J Bacteriol 148:265–273
Greer J (1990) Comparative modeling methods: application to the family of the mammalian serine proteases. Proteins Struc Funct Genet 7:317–334
Grimberg J, Maguire S, Belucio L (1989) A simple method for preparation of plasmid and chromosomal E. coli DNA. Nucleic Acids Res 17:8893
Grimm B (1990) Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde amino transferase. Proc Natl Acad Sci USA 87:4169–4173
Heijne G von (1985) Signal sequences. The limits of variation. J Mol Biol 184:99–105
Kanatani A, Masuda T, Shimoda T, Misoka F, Lin XS, Yoshimoto T, Tsuru D (1991) Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme. J Biochem 110:315–320
Kobayashi K, Lin LW, Yeadon JE, Klickstein LB, Smith JA (1989) Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase. J Biol Chem 264:8892–8899
Kraut J (1977) Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem 46:331–358
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Messing J, Gronenborn B, Müller-Hill B, Hofschneider PH (1977) Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci USA 74:3642–3646
Misumi Y, Hayashi Y, Arakawa F, Ikehara Y (1992) Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface. Biochim Biophys Acta 1131:333–336
Mitta M, Asada K, Uchimura Y, Kimizuka F, Kato J, Sakiyama F, Tsunasawa S (1989) The primary structure of porcine liver acylamino acid-releasing enzyme deduced from cDNA sequence. J Biochem 106:548–551
Morihara K (1987) Using proteases in peptide synthesis. TIBTECH 5:164–170
Moriyama A, Nakanishi M, Sakaki M (1988) Porcine muscle prolyl endopeptidase and its endogenous substrates. J Biochem 104:112–117
Naylor SI, Marshall A, Hensel C, Martinez PF, Holley B, Sakaguchi AY (1989) The DNS15S2 locus at 3p21 is transcribed in normal lung and small cell lung cancer. Genomics 4:353–361
Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biochem 240:3685–3692
Ogata S, Misumi Y, Ikehara Y (1989) Primary structure of rat liver dipeptidyl peptidase IV deduced from its cDNA and identification of the NH2-terminal signal sequence as the membrane-anchoring domain. J Biol Chem 264:3596–3601
Orlowski M, Wilk, Pearce S, Wilk S (1979) Purification and properties of a prolyl endopeptidase from rabbit brain. Neurochem 33:461–465
Osborn MJ, Gander JE, Parisi E, Carson J (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247:3962–3972
Polgár L (1992) Structural relationship between lipases and peptidases of prolyl oligopeptidase family. FEBS 311:281–284
Rawlings ND, Polgar L, Barrett AJ (1991) A new family of serine-type peptidase related to prolyl oligopeptidases. Biochem J 279:907–911
Rennex D, Hemmings BA, Hofsteenge J, Stone SR (1991) cDNA cloning of porcine brain endopeptidase and identification of the active-site seryl residue. Biochemistry 30:2195–2203
Roberts CJ, Pohlig, Rothman JH, Stevens TH (1989) Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole. J Cell Biol 108:1363–1373
Roggenkamp R, Kustermann-Kuhn B, Hollenberg CP (1981) Expression and processing of the bacterial β-lactamase in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 78:4466–4470
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Sanger F, Micklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sattar AKM, Yamamoto N, Yoshimoto T, Tsuru D (1990) Purification and characterization of an extracellular prolyl endopeptidase from Aspergillus bisporus. J Biochem 107:256–261
Shively JE (1986) Methods of protein microcharacterization. Humana Press, Clifton N.J.
Siezen RJ, Vos WM de, Leunissen JAM, Dijkstra BW (1991) Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Eng 7:719–737
Sommer J (1992) Synthesis of mammalian prolylendopeptidase in E. coli and analysis of the recombinant protein. Biochim Biophys Acta (in press)
Stone SR, Rennex D, Wikstrom P, Shaw E, Hofsteenge J (1991) Inactivation of prolyl endopeptidase by a peptidiyl-chloromethane. Kinetics of inactivation and identification of sites of modification. Biochem J 276:837–840
Stueber D, Ibrahim I, Cutler D, Doberstein B, Bujard H (1984) A novel in vitro transcrition-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J 3:3143–3148
Studier FW (1973) Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol 79:237–248
Svendsen I, Breddam K (1992) Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis. Eur J Biochem 204:165–171
Szwajcer-Dey E, Rasmus J, Meldac M, Breddam K (1992) Proline-specific endopeptidase from microbial sources: isolation of an enzyme from Xanthomonas sp. J Bacteriol 8:2454–2459
Tabor S, Richardson C (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Yoshimoto T, Walter R, Tsuru D (1980) Proline-specific endopeptidase from Flavobacterium, purification and properties. J Biol Chem 10:4786–4792
Yoshimoto T, Sattar AKM, Hirose W, Tsuru D (1987) Studies on prolyl endopeptidase from carrot (Daucus carota): purification and enzymatic properties. Biochim Biophys Acta 916:29–37
Yoshimoto T, Kanatani A, Shimoda T, Inaoka T, Kobubo T, Tsuru D (1991) Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene. J Biochem 110:873–878
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Diefenthal, T., Dargatz, H., Witte, V. et al. Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein. Appl Microbiol Biotechnol 40, 90–97 (1993). https://doi.org/10.1007/BF00170434
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DOI: https://doi.org/10.1007/BF00170434