Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase

Mierau, Igor; Tan, Paris S. T.; Haandrikman, Alfred J.; Mayo, Balthasar; Kok, Jan; Leenhouts, Kees; Konings, Wil N.; Venema, Gerard

doi:10.1128/jb.175.7.2087-2096.1993

Cited by 86 publications

(48 citation statements)

References 72 publications

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“…4B. The anti-PepO antibodies used also recognize a second homologous endopeptidase (PepO2), which is somewhat larger than PepO and specified elsewhere on the chromosome (Mierau et al 1993). As is clear from Fig.…”

Section: Gene Replacement In¸ Lactismentioning

confidence: 84%

“…In addition to the mutations described, the pORI system has been used to mutate the dtp¹, pepO, pep¹ and acmA genes of¸. lactis (Mierau et al 1993(Mierau et al , 1994Hagting et al 1994;Buist et al 1995).…”

Section: Comments On the Methodsmentioning

confidence: 99%

“…The lactococcal endopeptidase gene pepO was chosen as a chromosomal target to examine whether this promoter would be strong enough to express a gene downstream of the insertion point. pepO is normally transcribed from promoter P upstream of oppA, the gene immediately preceding pepO (Mierau et al 1993;Tynkkynen et al 1993; see Fig. 4A, line 3).…”

Section: Gene Replacement In¸ Lactismentioning

confidence: 99%

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A general system for generating unlabelled gene replacements in bacterial chromosomes

et al. 1996

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A general system for generating unlabelled gene replacements in bacterial chromosomes Leenhouts, K.; Buist, Girbe; Bolhuis, A.; Berge, A. ten; Kiel, Jan; Mierau, I.; Dabrowska, M.; Venema, G.; Kok, Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date: 1996Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Leenhouts, K., Buist, G., Bolhuis, A., Berge, A. T., Kiel, J., Mierau, I., ... Kok, J. (1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Molecular and General Genetics MGG, 253(1-2), 217-224. DOI: 10.1007/s004380050315 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Abstract A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using¸actococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

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Section: Gene Replacement In¸ Lactismentioning

confidence: 84%

Section: Comments On the Methodsmentioning

confidence: 99%

Section: Gene Replacement In¸ Lactismentioning

confidence: 99%

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A general system for generating unlabelled gene replacements in bacterial chromosomes

et al. 1996

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“…In the recently determined nucleotide sequence of the endopeptidase gene, pep0, from L. lactis subsp. cremoris P8-2-47 (Mierau et al, 1993), the translation start site corresponds to the codon specifying the N-terminus of the mature protein, showing that no signal sequence characteristic of membrane-translocated proteins is present ; this provides further confirmation of the intracellular location.…”

Section: Cellular Location Of Endopeptidase Activitymentioning

confidence: 71%

“…This N-terminal sequence is identical t o that predicted from the nucleotide sequence for the endopeptidase from L. lactis subsp. cremorir P8-2-47 (Mierau et al, 1993). However, it shows almost no homology with the N-terminal sequence determined for LEP-I1 from L. lactis subsp.…”

Section: N-terminal Sequencementioning

confidence: 89%

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

Pritchard

Freebairn²,

Coolbear³

1994

Microbiology

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An endopeptidase has been purified from Lactococcus lactis subsp. crernoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,l O-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor plchloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl f luoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. T w o of the oligopeptides produced during the early stages of & casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic s ys tem .

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Neprilysin

Dale¹,

Oefner²

2004

Handbook of Metalloproteins

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Neprilysin (NEP), a thermolysin‐like zinc metalloendopeptidase, is the major enzyme involved in the metabolic inactivation of a number of bioactive peptides including the enkephalins, substance P, endothelin, bradykinin, and the atrial natriuretic factor. NEP is an integral plasma membrane endopeptidase of the M13 family of zinc peptidases. Other related mammalian NEP‐like enzymes include the endothelin‐converting enzymes (ECE‐1 and ECE‐2), KELL and PEX. A number of novel mammalian homologues of NEP have also recently been described. Owing to the physiological importance of NEP in the modulation of nociceptive and pressor responses, there is considerable interest in inhibitors of this enzyme as novel analgesics and antihypertensive agents. Here we provide an overview of this pharmacologically important protein protease family.

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Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase

Cited by 86 publications

References 72 publications

A general system for generating unlabelled gene replacements in bacterial chromosomes

A general system for generating unlabelled gene replacements in bacterial chromosomes

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

Neprilysin

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