Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases

Sánchez-Puelles, José María; Sanz, Jesús M.; García, José Luis Rueda; Garcı́a, Ernesto

doi:10.1016/0378-1119(90)90207-8

Cited by 107 publications

(97 citation statements)

References 20 publications

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“…Immobilization and purification of a hybrid protein ChoBD -b-galactosidase Plasmid pCEl7 contains the 3' moiety of the lytA gene of S. pneumoniae downstream of a modified E. coli lipoprotein promoter (1ppp-5) and the lactose promoter/operator region (lacP0) and, consequently, C-LYTA can be synthesized upon the addition of lac inducers [13]. We previously reported that deletions or insertions in the C-terminus of the pneumococcal amidase did not affect the capacity of the enzyme to bind to choline-containing supports [24].…”

Section: Results and Dicussionmentioning

confidence: 99%

“…[18], pUC9 [16], pMC1043 [15], pMC931 [15], pMC1871 [15], pCMl [13], pCE17 [33] and pJCP191 [19]. The bacteriophage M13tg131 (Amersham) was used in the construction of plasmid pCUZ1.…”

Section: The Plasmids Used Were Piniii-(lppp-5-lac)-a3mentioning

confidence: 99%

“…The lytA gene encoding the LYTA amidase and the cpll gene encoding the CPLl lysozyme have been cloned and sequenced , showing a modular organization [12]. Recently, we have cloned the gene fragments coding for the homologous C-terminal choline-binding domains (ChoBD) of the LYTA amidase (C-LYTA) and the CPLl lysozyme (C-CPL1) [13]. Both polypeptides exhibit strong affinity for matrices containing choline or some choline analogues such as DEAE [14].…”

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confidence: 99%

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Immobilization and single‐step purification of fusion proteins using DEAE‐cellulose

Sánchez-Puelles

Sanz

Garcı́a

et al. 1992

European Journal of Biochemistry

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We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding-domain -P-galactosidase chimera, which can be purified by this procedure and shows a high P-galactosidase activity when immobilized in the column. A vector plasmid, pCUZl, containing the Ippp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-~-~-galactoside procedure.

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Section: Results and Dicussionmentioning

confidence: 99%

“…[18], pUC9 [16], pMC1043 [15], pMC931 [15], pMC1871 [15], pCMl [13], pCE17 [33] and pJCP191 [19]. The bacteriophage M13tg131 (Amersham) was used in the construction of plasmid pCUZ1.…”

Section: The Plasmids Used Were Piniii-(lppp-5-lac)-a3mentioning

confidence: 99%

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confidence: 99%

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Immobilization and single‐step purification of fusion proteins using DEAE‐cellulose

Sánchez-Puelles

Sanz

Garcı́a

et al. 1992

European Journal of Biochemistry

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“…Expression and Purification of Cpl-1 Endolysin-The native Cpl-1 lysozyme and its Cpl-1 E94Q mutant variant were expressed in Escherichia coli DH1 (pCIP100) and E. coli DH1 (pCOB7) cells, respectively, and purified from the crude extracts as described (14).…”

Section: Methodsmentioning

confidence: 99%

Elucidation of the Molecular Recognition of Bacterial Cell Wall by Modular Pneumococcal Phage Endolysin CPL-1

Pérez‐Dorado

Campillo

Monterroso

et al. 2007

Journal of Biological Chemistry

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Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.Streptococcus pneumoniae is one of the most common and important human pathogens, which causes serious life-threatening diseases such as acute otitis media, pneumonia, sepsis, and meningitis. Pneumococcal infections are associated with high morbidity and mortality, especially among children, the elderly, and the immune-depressed patients. The widespread emergence of antibiotic resistance and the lack of highly effective pneumococcal vaccines against all serotypes of this organism give urgency to elucidation of the molecular processes involved in its pathogenicity (1, 2).The peptidoglycan (PG) 3 scaffold of the bacterial cell wall is a repeating GlcNAc-N-acetylmuramic (MurNAc) disaccharide (GlcNAc-(␤-1,4)-MurNAc) unit having a pentapeptide attached to the D-lactyl moiety of each MurNAc unit. All known pneumococcal bacteriophages encode an amidase or a lysozyme, which hydrolyzes the PG at the final stage of the phage reproductive cycle, leading to bacterial cell lysis. These enzymes, known collectively as endolysins, have been shown to be highly efficient in killing pneumococci in vitro and can eradicate this organism from the upper respiratory tract or from the bloodstream of mice (3, 4) acting as new antimicrobial agents (i.e. enzybiotics). In addition, Cpl-1 lysin and Pal amidase encoded by phage Dp-1 act in a synergistic manner in a sepsis mouse model (5); this synergy has also been confirmed in in vitro experiments with Cpl-1 and penicillin or gentamicin (6). Very recently, the creation of a new animal model of otitis media has been reported (7). Using this new mouse model, it has been demonstrated that Cpl-1 could eliminate colonization with S. p...

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“…1) and it proved possible to use this resin for affinity chromatography in a similar manner to the approach previously reported using DEAE resin (Sanchez-Puelles et al, 1990). Protein previously purified by Ni-NTA affinity was dialysed into 50 mM Tris-HCl pH 7.5, 150 mM NaCl and loaded onto a column packed with 50 ml Q-Sepharose resin.…”

Section: Purificationmentioning

confidence: 96%

Overexpression, purification and crystallization of a choline-binding protein CbpI fromStreptococcus pneumoniae

et al. 2006

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The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni-NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å , = = = 90 . The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V M = 2.77 Å 3 Da À1 ) and shows a diffraction limit of 3.5 Å .

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Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases

Cited by 107 publications

References 20 publications

Immobilization and single‐step purification of fusion proteins using DEAE‐cellulose

Immobilization and single‐step purification of fusion proteins using DEAE‐cellulose

Elucidation of the Molecular Recognition of Bacterial Cell Wall by Modular Pneumococcal Phage Endolysin CPL-1

Overexpression, purification and crystallization of a choline-binding protein CbpI fromStreptococcus pneumoniae

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