Comparison of different cell disruption methods and cell extractant buffers for recombinant bromelain expressed in E.coli BL21-A1
The often-encountered problem such as protein degradation has driven various methods of cell lysis in obtaining recombinant protein post fermentation. In this paper, we compare methods such as homogenization, sonication, sonication with lysozyme and chemical lysis using B-PER reagent with lysozym...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Universiti Teknologi Malaysia
2015
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/46349/ http://irep.iium.edu.my/46349/ http://irep.iium.edu.my/46349/1/Cell_disruption.pdf |
| Summary: | The often-encountered problem such as protein degradation has driven various
methods of cell lysis in obtaining recombinant protein post fermentation. In this
paper, we compare methods such as homogenization, sonication, sonication with
lysozyme and chemical lysis using B-PER reagent with lysozyme to extract the
recombinant bromelain from E. coli BL21-AI. The sonication process is found to be
the most effective in releasing recombinant bromelain without any pre-treatment.
To obtain the high quality of protein from sonication method, the influence of
different extractant buffer was investigated including phosphate buffer saline (PBS),
PBS containing cysteine and EDTA (PBS-CE), and sodium phosphate buffer
containing cysteine and EDTA (EB-CE). The highest specific enzyme activity was
obtained when it was extracted with EB-CE buffer. Under sodium dodecyl sulfate
polyacrylamide gel electrophoresis, the recombinant bromelain showed protein
band at 55kDa. In conclusion, the sonication method with extractant buffer
containing 100mM phosphate buffer pH7.0 with 15 mM cysteine and 2 mM EDTA
(EB-CE) was shown to give high specific activity of recombinant bromelain. |
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