Difference between revisions of "Part:BBa K1166002"
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==Introduction== | ==Introduction== | ||
| − | The alpha-hemolysin system is one of the best-studied type 1 secretion systems (T1SS) of ''E. coli''. In T1SS the secretion occurs in a single step directly from the cytosol to the extracellular medium. The secretory machinery of the alpha-hemolysin system consists of three proteins: HlyB, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein (Su, et al., 2012). | + | The alpha-hemolysin system is one of the best-studied type 1 secretion systems (T1SS) of ''E. coli''. In T1SS the secretion occurs in a single step directly from the cytosol to the extracellular medium. The secretory machinery of the alpha-hemolysin system consists of three proteins: HlyB, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein (Su, et al., 2012). The natural substrate HlyA can be secreted because it contains a C-terminal signal and it has been shown that proteins with C-terminally fused HlyA signal sequence can also be recognized by the HlyB-HlyD-TolC translocator (Gentschev, et al., 2002). |
==Usage== | ==Usage== | ||
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Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. (2012). Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 11:8 | Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. (2012). Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 11:8 | ||
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| + | Gentschev I, Dietrich G, Goebel W. (2002). The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol. 10(1):39-45. | ||
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Revision as of 19:48, 27 September 2013
HlyA-tag+Secretion system
Introduction
The alpha-hemolysin system is one of the best-studied type 1 secretion systems (T1SS) of E. coli. In T1SS the secretion occurs in a single step directly from the cytosol to the extracellular medium. The secretory machinery of the alpha-hemolysin system consists of three proteins: HlyB, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein (Su, et al., 2012). The natural substrate HlyA can be secreted because it contains a C-terminal signal and it has been shown that proteins with C-terminally fused HlyA signal sequence can also be recognized by the HlyB-HlyD-TolC translocator (Gentschev, et al., 2002).
Usage
This is a device that allows a protein to be secreted by means of the alpha-hemolysin secretion system in E. coli. It’s designed so that the only thing that you have to do is to assemble your protein part in the device via the biofusion standard (BBF RFC 23), (Cut the device with EcoRI and XbaI, cut your protein part with EcoRI and SpeI, mix & ligate). This procedure will leave your part in frame with a signal peptide.
Note: It’s important that your part doesn’t contain a stop codon nor a terminator since your protein will be fused at the C-terminus.
References
Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. (2012). Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 11:8
Gentschev I, Dietrich G, Goebel W. (2002). The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol. 10(1):39-45.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1550
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1489
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1324
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1306