Proteomic study on the recombinant strain of Escherichia coli
| Autor: | Cheng-Chung Hsien, 鄭仲賢 |
|---|---|
| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 The study and application of cellular proteins is the main field for research in the post-genome era proteomics provides a powerful tool for understanding the roles of proteins in an organism. Escherichia coli is most frequently as a host cell for the expression of recombinant proteins. Databases related to Escherichia coli updating and approaching to be complete. This study is to analyze the differences of the proteomes of the host cell Escherichia coli BL21, induced and non-induced recombinant Escherichia coli BL21 (including a recombinant plasmid). Alternatively, we used silver nitrate and silver diammine protocols to detect proteins and finding on the two-dimensional electrophoresis (2-DE) gels. We could detect more proteins by using silver diammine protocol. However, in contrast to silver nitrate protocol, the silver diammine protocol was time consuming and costly. Also, vertical and horizontal streaks were more observable and subject to interfere software analysis. The silver nitrate was thus superior. As the protein redissolving time is increasing to five minutes, the number of spots appeared on the gel was not less than that by using the method of silver diammine. On 2-DE gels, we have identified two protein spots: gapA (glyceraldehyde 3-phosphatedehydrogenase，MW 35.2K Da, pI 6.58 ) and dnaK (Chaperon Hsp70，MW 69K Da, pI 4.83), by using LC-MS/MS. Protein gapA is one of the housekeeping proteins and can serve as the standard for the normalization of proteins for quantitative analysis. We did not find the target protein: GST-nanA-Arg5 on both the 2-DE gels of proteins from induced and non-induced recombinant Escherichia coli BL21 cells, even when the amounts of proteins loaded to gels was increased to 500 μg. However, the presence of the target protein on the 2-DE gel of the purified protein was obvious, confirmed by western blotting. The result from western blotting implied that there were several proteins associated with GST tag. We need to investigate more detail on why the target protein in the original extracted solution did not show up on the 2-DE gel and to carry out the experiments of western blotting. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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