Rapid purification of bacteriophage endolysin TSPphg and its exogenous treatment could act as an alternative bacterial cell disruption method

Wu, Feng; Ji, Xinyu; Chen, Meishan; Guo, Jun; Deng, Xian-Yu; Lü, Lin

doi:10.1016/j.pep.2018.03.014

Cited by 8 publications

(8 citation statements)

References 17 publications

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“…The highly efficient expression of TSPphg in the host E. coli BL21 cells and its rapid purification using the special thermolysis method were already described in detail in our previous publication [15]. Briefly, TSPphg gene was amplified by PCR with gene-specific primers from the phage TSP4 genome (forward: 5'-CATGCCATGGCAATGCGTCTACCGACTAAGAC-3' and reverse: 5'-CCGCTCGAGTTTACCTCCTAGCAACTTGG-3').…”

Section: Production and Purification Of Recombinant Protein Tspphgmentioning

confidence: 99%

“…The natural DNA sequence of the TSPphg gene in the phage TSP4 genome was cloned by PCR. Overexpression and rapid purification of heat-stable TSPphg protein from the host E. coli BL21 cells were previously described in our publication [15], and there is no difference in the sequence between recombinant and wild-type TSPphg lysin. The purified TSPphg lysin used in this study was confirmed by 12% SDS-PAGE ( Figure 1C).…”

Section: Identification Of Tspphg By Sequencing and Annotation Of Phamentioning

confidence: 99%

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TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

et al. 2020

Viruses

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New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 µg/mL TSPphg treatment at 37 • C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.

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Section: Production and Purification Of Recombinant Protein Tspphgmentioning

confidence: 99%

Section: Identification Of Tspphg By Sequencing and Annotation Of Phamentioning

confidence: 99%

TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

et al. 2020

Viruses

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“…After induction, cells were centrifugated at 1000× g for 10 min at 4 • C, and when necessary, the pellets can be stored in a freezer at −80 • C until further use. The ultrasonication was carried out as described previously [19] with a Sonics VCX500 (Sonics & Materials Inc., Newtown, CT, USA) and the acoustic power was set as 120 W (0.5 s on and 0.5 off). After sonication, the cell debris was pelleted by centrifuging at 12,000× g for 10 min at 4 • C. For Ni-affinity purification of the recombinant protein, a HisTrap™ HP column was employed following the manufacturer's guidelines (GE Healthcare, Pittsburgh, PA, USA), and the target protein was obtained by eluting with imidazole (from 50 to 500 mM).…”

Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

“…The protein concentrations were determined quantitatively using the routine Bradford measurement, as described previously [19]. Bovine serum albumin (BSA) in a graded concentration series were used as the known standards, and the final OD 595 was measured with a spectrophotometer (V729, YOKE, Shanghai, China).…”

Section: Determination Of Protein Concentrationsmentioning

confidence: 99%

Design, Overproduction and Purification of the Chimeric Phage Lysin MLTphg Fighting against Staphylococcus aureus

Liu

Deng

et al. 2020

Processes

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With the increasing spread of multidrug-resistant bacterial pathogens, it is of great importance to develop alternatives to conventional antibiotics. Here, we report the generation of a chimeric phage lysin, MLTphg, which was assembled by joining the lysins derived from Meiothermus bacteriophage MMP7 and Thermus bacteriophage TSP4 with a flexible linker via chimeolysin engineering. As a potential antimicrobial agent, MLTphg can be obtained by overproduction in Escherichia coli BL21(DE3) cells and the following Ni-affinity chromatography. Finally, we recovered about 40 ± 1.9 mg of MLTphg from 1 L of the host E. coli BL21(DE3) culture. The purified MLTphg showed peak activity against Staphylococcus aureus ATCC6538 between 35 and 40 °C, and maintained approximately 44.5 ± 2.1% activity at room temperature (25 °C). Moreover, as a produced chimera, it exhibited considerably improved bactericidal activity against Staphylococcus aureus (2.9 ± 0.1 log10 reduction was observed upon 40 nM MLTphg treatment at 37 °C for 30 min) and also a group of antibiotic-resistant bacteria compared to its parental lysins, TSPphg and MMPphg. In the current age of growing antibiotic resistance, our results provide an engineering basis for developing phage lysins as novel antimicrobial agents and shed light on bacteriophage-based strategies to tackle bacterial infections.

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“…SDS-PAGE was conducted as described previously [16]: The samples were first boiled at 95°C for 5 min after the addition of 20% (v/v) 5× SDS loading buffer (250 mM Tris-HCl pH6.8, 10% SDS, 0.5% BPB, 50% glycerol, 5% 2-ME). Electrophoresis was then performed in the 0.75 mm-thick SDS-PAGE gel containing 12% polyacrylamide in Tris-glycine running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) at 80-100 V, and proteins were further visualized by staining with Coomassie R250 solution (0.1% Coomassie R250, 25% isopropanol, 10% acetic acid).…”

Section: Sds-pagementioning

confidence: 99%

Using enzymatic hydrolyzate as new nitrogen source for L-tryptophan fermentation by E.coli

Zhang

Liu

et al. 2019

Bioengineered

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This study presents new methods for hydrolyzing bacterial cell in cyclic utilization of waste bacterial cell for L-tryptophan production by fermentation. Using enzymatic hydrolysis of the pre-treated bacterial cells which were collected from an L-tryptophan fermentation broth, trypsin was selected as the optimal protease for hydrolyzing the bacterial cell. The optimum conditions for hydrolysis were determined by the orthogonal test. Hydrolyzate was then dealt with a compound protease to further increase its content of free amino acids. With the optimum conditions of pH = 8, temperature of 37°C, treatment time of 6 h, and E/S of 4%, the final content of free amino acids in the hydrolyzate was 500.61 mg/g. The hydrolyzate and the yeast extract were added to the medium at the proportion of 1:1, which served as an organic nitrogen source for L-tryptophan production by fermentation. The production of L-tryptophan was 53.87 g/L, and the highest biomass was 53.45 g/L. As an organic nitrogen source, this hydrolyzate satisfies the requirements for L-tryptophan production by fermentation.

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Rapid purification of bacteriophage endolysin TSPphg and its exogenous treatment could act as an alternative bacterial cell disruption method

Cited by 8 publications

References 17 publications

TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

Design, Overproduction and Purification of the Chimeric Phage Lysin MLTphg Fighting against Staphylococcus aureus

Using enzymatic hydrolyzate as new nitrogen source for L-tryptophan fermentation by E.coli

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