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Team:Grinnell/Project/RsaA
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<p>RsaA is the surface-layer (S-layer) protein produced by the gram-negative bacteria <i>Caulobacter crescentus</i>. It covers the surface of the cell in a 2-D hexameric crystal and accounts for 10-12% of total protein synthesis by the cell (<a href="https://2011.igem.org/Team:Grinnell/Attributions#Awram">Awram 1998</a>). Key regions of the protein include the N-terminal region, which is necessary for binding of the protein to the surface of the cell, and the C-terminal region, which acts as a secretion tag (<a href="https://2011.igem.org/Team:Grinnell/Attributions#Bingle">Bingle 2000</a>). The secretion pathway for RsaA is a robust ATP-dependent type I secretion pathway. These features of RsaA and its secretion pathway allows us to take advantage of it by creating chimeric proteins with the C-terminal secretion tag. These proteins should then be expressed and secreted in <i>Caulobacter</i> in relatively high yield. Another advantage of this is that <i>Caulobacter</i> secretes very few proteins, so isolation of the desired protein from liquid culture supernatant should be relatively easy.</p> | <p>RsaA is the surface-layer (S-layer) protein produced by the gram-negative bacteria <i>Caulobacter crescentus</i>. It covers the surface of the cell in a 2-D hexameric crystal and accounts for 10-12% of total protein synthesis by the cell (<a href="https://2011.igem.org/Team:Grinnell/Attributions#Awram">Awram 1998</a>). Key regions of the protein include the N-terminal region, which is necessary for binding of the protein to the surface of the cell, and the C-terminal region, which acts as a secretion tag (<a href="https://2011.igem.org/Team:Grinnell/Attributions#Bingle">Bingle 2000</a>). The secretion pathway for RsaA is a robust ATP-dependent type I secretion pathway. These features of RsaA and its secretion pathway allows us to take advantage of it by creating chimeric proteins with the C-terminal secretion tag. These proteins should then be expressed and secreted in <i>Caulobacter</i> in relatively high yield. Another advantage of this is that <i>Caulobacter</i> secretes very few proteins, so isolation of the desired protein from liquid culture supernatant should be relatively easy.</p> | ||
| - | <p>There is a limit on the effectiveness of expression of this type of recombinant protein. The main limiting factors are the size of the protein joined to the C-terminal of RsaA (smaller protein regions are better expressed and secreted) and the codons used in the gene in question (<i>Caulobacter</i> has a GC rich genome). In addition to addressing these issues, use of a strong constitutive promoter can help. In <i>Caulobacter</i> using the P<sub>rsaA</sub> promoter is effective, and | + | <p>There is a limit on the effectiveness of expression of this type of recombinant protein. The main limiting factors are the size of the protein joined to the C-terminal of RsaA (smaller protein regions are better expressed and secreted) and the codons used in the gene in question (<i>Caulobacter</i> has a GC rich genome). In addition to addressing these issues, use of a strong constitutive promoter can help. In <i>Caulobacter</i> using the P<sub>rsaA</sub> promoter is effective, and should become more effective if native RsaA production is turned off.</p> |
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Latest revision as of 04:03, 29 September 2011
RsaA
RsaA is the surface-layer (S-layer) protein produced by the gram-negative bacteria Caulobacter crescentus. It covers the surface of the cell in a 2-D hexameric crystal and accounts for 10-12% of total protein synthesis by the cell (Awram 1998). Key regions of the protein include the N-terminal region, which is necessary for binding of the protein to the surface of the cell, and the C-terminal region, which acts as a secretion tag (Bingle 2000). The secretion pathway for RsaA is a robust ATP-dependent type I secretion pathway. These features of RsaA and its secretion pathway allows us to take advantage of it by creating chimeric proteins with the C-terminal secretion tag. These proteins should then be expressed and secreted in Caulobacter in relatively high yield. Another advantage of this is that Caulobacter secretes very few proteins, so isolation of the desired protein from liquid culture supernatant should be relatively easy.
There is a limit on the effectiveness of expression of this type of recombinant protein. The main limiting factors are the size of the protein joined to the C-terminal of RsaA (smaller protein regions are better expressed and secreted) and the codons used in the gene in question (Caulobacter has a GC rich genome). In addition to addressing these issues, use of a strong constitutive promoter can help. In Caulobacter using the PrsaA promoter is effective, and should become more effective if native RsaA production is turned off.
