Genetic and Physical Map of the pLAFR1 Vector

Vanbleu, Els; Marchal, Kathleen; Vanderleyden, Jozef

doi:10.1080/10425170410001723949

Cited by 15 publications

(7 citation statements)

References 30 publications

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“…The protein was modified to carry two N -glycosylation sequons (N262 and N404), a C-terminal His tag and a signal peptide sequence from the E. coli protein DsbA fused to the N-terminal to localize ExoA to the periplasm of the E. coli host cell [15]. The F. tularensis O-antigen coding region was subcloned from pGAB1 into the unique Eco RI site of the low copy vector pLAFR1 [33] to yield the vector pGAB2. The plasmids pGAB2, pGVXN114 and pGVXN150 containing the O-antigen, CjPglB and ExoA, respectively, were transformed into E. coli strain CLM24 that has a deletion in the waaL gene rendering the E. coli host strain ligase negative and unable to transfer UndPP-linked glycan to lipid A, therefore generating a lipid-linked substrate specific for CjPglB.…”

Section: Resultsmentioning

confidence: 99%

Exploitation of bacterialN-linked glycosylation to develop a novel recombinant glycoconjugate vaccine againstFrancisella tularensis

et al. 2013

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Glycoconjugate-based vaccines have proved to be effective at producing long-lasting protection against numerous pathogens. Here, we describe the application of bacterial protein glycan coupling technology (PGCT) to generate a novel recombinant glycoconjugate vaccine. We demonstrate the conjugation of the Francisella tularensis O-antigen to the Pseudomonas aeruginosa carrier protein exotoxin A using the Campylobacter jejuni PglB oligosaccharyltransferase. The resultant recombinant F. tularensis glycoconjugate vaccine is expressed in Escherichia coli where yields of 3 mg l−1 of culture were routinely produced in a single-step purification process. Vaccination of BALB/c mice with the purified glycoconjugate boosted IgG levels and significantly increased the time to death upon subsequent challenge with F. tularensis subsp. holarctica. PGCT allows different polysaccharide and protein combinations to be produced recombinantly and could be easily applicable for the production of diverse glycoconjugate vaccines.

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Section: Resultsmentioning

confidence: 99%

Exploitation of bacterialN-linked glycosylation to develop a novel recombinant glycoconjugate vaccine againstFrancisella tularensis

et al. 2013

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“…Recombinant EPA expressed from pGVXN150 contains two engineered N-glycosylation sites (N262 and N398) and is fused to a hexahistidine tag at the C-terminal end and to the DsbA signal peptide at the N-terminal end (Sec-dependent secretion to the periplasm). Tetracycline-selectable, low copy number plasmid pGVXN64 was used for biosynthesis of O antigen polysaccharides of S. dysenteriae serotype 1. pGVXN64 (origin of replication: IncPα) was constructed by insertion of an 11 kb Bam HI fragment of pSDM7 [18] containing the S. dysenteriae rfp and rfb gene clusters into the Bam HI site of pLAFR1 [19,20]. The rfp and rfb gene clusters encode glycosyltransferases and polymerases required for the synthesis of undecaprenyl-pyrophosphate-linked Shigella O1 polysaccharides [18] and were expressed from their native (constitutive) promoters in pGVXN64.…”

Section: Methodsmentioning

confidence: 99%

Production of glycoprotein vaccines in Escherichia coli

Kowarik²,

et al. 2010

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BackgroundConjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.ResultsTwo different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.ConclusionsThe presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

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“…This is especially true for plasmid replicons. The two most commonly used plasmid replicons for P. aeruginosa research are pRK2 (pRP4) in the pLAFR series of vectors (Friedman et al, 1982, McIver et al, 1993, Vanbleu et al, 2004) and the pRO1610 replicon that is designated as the Pseudomonas stabilizing fragment (SF) (Olsen et al, 1982). The SF has been incorporated into the pUC series of E. coli plasmid cloning vectors to generate P. aeruginosa-E. coli shuttle vectors designated as the pUCP series of vectors (Schweizer, 1991, West et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

Silo-Suh

Elmore

Ohman

et al. 2009

Journal of Microbiological Methods

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In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF ts1 . The mutations that resulted in a temperature-sensitive phenotype in mSF ts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42°C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 hours of growth. In order to facilitate its utilization, the mSF ts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF ts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.

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Genetic and Physical Map of the pLAFR1 Vector

Cited by 15 publications

References 30 publications

Exploitation of bacterialN-linked glycosylation to develop a novel recombinant glycoconjugate vaccine againstFrancisella tularensis

Exploitation of bacterialN-linked glycosylation to develop a novel recombinant glycoconjugate vaccine againstFrancisella tularensis

Production of glycoprotein vaccines in Escherichia coli

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

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