Optimization of BLyS production and purification from Escherichia coli

Laird, Michael W.; Sampey, Gavin C.; Johnson, Kelly E.; Zukauskas, David; Pierre, Jennifer L.; Hong, June S; Cooksey, Bridget A.; Li, Yuling; Galperina, Olga; Karwoski, Jeffrey D.; Burke, Robert N.

doi:10.1016/j.pep.2004.10.006

Cited by 15 publications

(11 citation statements)

References 31 publications

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“…Plasmid constructs and DNA manipulation. Bacteriophage vB_SauS-phiIPLA88 gene orf58 (open reading frame 58) encoding HydH5 (GenBank accession number ACJ64586) was codon optimized (orf58-opt) based on the E. coli codon usage (23) and commercially synthesized (Genescript, Piscataway, NJ). Then, orf58-opt was synthesized with a 5= NdeI and a 3= XhoI restriction enzyme site and the resultant fragment was subcloned between the NdeI and XhoI sites in the multicloning site of the inducible expression vector pET21a (EMD Biosciences, San Diego, CA), which introduces a C-terminal 6ϫHis tag.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Rodríguez-Rubio

Martínez

Rodrı́guez

et al. 2012

Appl Environ Microbiol

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ABSTRACTVirion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from theStaphylococcus aureusbacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against liveS. aureuscells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysedS. aureuscells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and humanS. aureusstrains, the methicillin-resistantS. aureus(MRSA) strain N315, and humanStaphylococcus epidermidisstrains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.

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Section: Methodsmentioning

confidence: 99%

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Rodríguez-Rubio

Martínez

Rodrı́guez

et al. 2012

Appl Environ Microbiol

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“…It enhances the humoral responses to both T cellindependent and T cell-dependent antigens. It has been cloned and expressed in both E. coli (Laird et al 2005) and African green monkey kidney fibroblast cells (COS-7) (Dai et al 2001) expression systems.…”

Section: Introductionmentioning

confidence: 99%

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Zhang

Tan

2006

Biotechnol Lett

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Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l(-1) and a productivity of 0.11 g l(-1) h(-1).

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“…cremoris MG1363 codon usage (Genescript) (17). The resulting DNA fragment was cloned into BamHI/PstI restriction sites from pNZ8020 to generate pNZ8020-SP Lcn972 -LysH5opt.…”

mentioning

confidence: 99%

Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

Rodríguez-Rubio

Gutiérrez

Martínez

et al. 2012

Appl Environ Microbiol

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ABSTRACTBacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded byStaphylococcus aureusphage vB_SauS-phi-IPLA88, was expressed and secreted inLactococcus lactisusing the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.

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Optimization of BLyS production and purification from Escherichia coli

Cited by 15 publications

References 31 publications

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

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