Application
Protein was extracted by Omni Sonic ruptor from E. coli to be used for SDS-PAGE
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작성일
2021-09-30 11:06
Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice
Mustafa et al. BMC Biotechnology (2018) 18:63,
> https://doi.org/10.1186/s12896-018-0461-y
Background:
Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world’s population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0–80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed.
Conclusions:
This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.
Keywords:
M. tuberculosis vaccine, Surface display vaccine, L. plantarum, In-trans approach
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Methods-Protein expression and extraction
Inoculation of E. coli Rossetta harbouring pRSF:ACERL or empty pRSF:Duet-1 (as negative control) into fresh TB medium was performed using overnight cultures and grown to an OD 600nm of 0.5. The recombinant E. coli were expressed for 6 h with 1 mM/mL IPTG at 30 °C with haking at 200 rpm. The cells were then harvested by centrifugation at 8000×g for 10 min at 4 °C. At this point, if required, the cells can be stored at -80 °C prior to protein extraction. For extraction, the cell pellets containing the protein of interest were prepared for sonication treatment using the Omni Ruptor 4000 (Omni International, USA) set to 40% power and pulsed for 8 min per sample. After that, the sonicated cell suspensions were centrifuged at 12000×g, for 20 min, at 4 °C. Both supernatant and pellet samples were isolated and further verified for the presence of the protein of interest via SDS-PAGE analysis before proceeding with the protein extraction and purification steps.
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Equipment:
Sonic Ruptor 4000 > 제품 상세정보 보러 가기
• 250μl에서 1L까지 처리 가능
• Titanium probes-쉬운 교환, 자동 튜닝
• 펄스 모드-온도 상승 제한
• 타이머: 0 ~ 15 minutes
• 0.01 micron 이하 emulsion 생성 가능
• 소음 저감 챔버 with clear plexiglass door
Applications:
• Nanoparticle dispersion
• Creating emulsions
• Cell disruption
• DNA shearing.
Tags:
Sonic Ruptor, bacteria, protein, E. coli, protein expression, SDS-PAGE
Mustafa et al. BMC Biotechnology (2018) 18:63,
> https://doi.org/10.1186/s12896-018-0461-y
Background:
Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world’s population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0–80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed.
Conclusions:
This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.
Keywords:
M. tuberculosis vaccine, Surface display vaccine, L. plantarum, In-trans approach
----------
Methods-Protein expression and extraction
Inoculation of E. coli Rossetta harbouring pRSF:ACERL or empty pRSF:Duet-1 (as negative control) into fresh TB medium was performed using overnight cultures and grown to an OD 600nm of 0.5. The recombinant E. coli were expressed for 6 h with 1 mM/mL IPTG at 30 °C with haking at 200 rpm. The cells were then harvested by centrifugation at 8000×g for 10 min at 4 °C. At this point, if required, the cells can be stored at -80 °C prior to protein extraction. For extraction, the cell pellets containing the protein of interest were prepared for sonication treatment using the Omni Ruptor 4000 (Omni International, USA) set to 40% power and pulsed for 8 min per sample. After that, the sonicated cell suspensions were centrifuged at 12000×g, for 20 min, at 4 °C. Both supernatant and pellet samples were isolated and further verified for the presence of the protein of interest via SDS-PAGE analysis before proceeding with the protein extraction and purification steps.
----------
Equipment:
Sonic Ruptor 4000 > 제품 상세정보 보러 가기
• 250μl에서 1L까지 처리 가능
• Titanium probes-쉬운 교환, 자동 튜닝
• 펄스 모드-온도 상승 제한
• 타이머: 0 ~ 15 minutes
• 0.01 micron 이하 emulsion 생성 가능
• 소음 저감 챔버 with clear plexiglass door
Applications:
• Nanoparticle dispersion
• Creating emulsions
• Cell disruption
• DNA shearing.
Tags:
Sonic Ruptor, bacteria, protein, E. coli, protein expression, SDS-PAGE
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