Difference between revisions of "Part:BBa K2114998"

Revision as of 00:32, 6 October 2016

pelB_aGFPnano

The discovery of camelid heavy-chain antibodies and their subsequent modification to single-domain antibodies also called nanobodies1,2 provide researchers with a wide range of tools including labeling methods for imaging, receptor modulation or therapeutic agents. They exhibit a small size of only 15 kDa and their easy structure enables a high-efficiency production in bacterial strains such as E. coli. The anti-GFP nanobody represents an established and well characterized variant of those proteins. In order to directly utilize their functionality, the purification of the over-expressed nanobody provides the advantage of acquiring a highly concentrated protein solution. The BioBrick in the iGEM registry BBa_K929104 contains the full sequence of the anti-GFP nanobody. However, the purification of the nanobody from a bacterial lysate requires its export into the periplasmatic space in order to avoid inclusion bodies and provide an oxidative environment which promotes the formation of the characteristic disulfide bond. The anti-GFP nanobody was provided to us by our supervisor Dr. Maximilian Ulbrich. To avoid the formation of inclusion bodies and increase the yield of purified nanobodies the pelB leader sequence was included at the N-terminal section of the nanobody. This sequence facilitates the export of the protein into the periplasmatic space.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 87
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 54
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2114998 short</partinfo>
-+++ TEST TEST TEST +++
-The discovery of camelid heavy-chain antibodies and their subsequent modification to single-domain antibodies also called nanobodies1,2 provide researchers with a wide range of tools including labeling methods for imaging, receptor modulation or therapeutic agents. They exhibit a small size of only 15 kDa and their easy structure enables a high-efficiency production in bacterial strains such as E. coli. The anti-GFP nanobody represents an established and well characterized variant of those proteins. In order to directly utilize their functionality, the purification of the over-expressed nanobody provides the advantage of acquiring a highly concentrated protein solution. The BioBrick in the iGEM registry BBa_K929104 contains the full sequence of the anti-GFP nanobody. However, the purification of proteins from a bacterial lysate involves a specific affinity tag by which they can be enriched. An anti-GFP nanobody variant containing a 10XHis-Tag and a TEV protease cleavage site was provided to us by our supervisor Dr. Maximilian Ulbrich. The His-Tag enables the purification by standard nickel columns while the TEV cleavage site allows the . To further avoid the formation of inclusion bodies and increase the yield of purified nanobodies an additional pelB leader sequence was included at the N-terminal section of the nanobody resulting in its transport into the periplasmatic space of the expressing E. coli. The pelB leader sequence  facilitates the export of the protein into the periplasmatic space and therefore ensures the proper folding of the nanobody by providing an oxidative environment which promotes the formation of the characteristic disulfide bond.
+The discovery of camelid heavy-chain antibodies and their subsequent modification to single-domain antibodies also called nanobodies1,2 provide researchers with a wide range of tools including labeling methods for imaging, receptor modulation or therapeutic agents. They exhibit a small size of only 15 kDa and their easy structure enables a high-efficiency production in bacterial strains such as E. coli. The anti-GFP nanobody represents an established and well characterized variant of those proteins. In order to directly utilize their functionality, the purification of the over-expressed nanobody provides the advantage of acquiring a highly concentrated protein solution. The BioBrick in the iGEM registry BBa_K929104 contains the full sequence of the anti-GFP nanobody. However, the purification of the nanobody from a bacterial lysate requires its export into the periplasmatic space in order to avoid inclusion bodies and provide an oxidative environment which promotes the formation of the characteristic disulfide bond. The anti-GFP nanobody was provided to us by our supervisor Dr. Maximilian Ulbrich. To avoid the formation of inclusion bodies and increase the yield of purified nanobodies the pelB leader sequence was included at the N-terminal section of the nanobody. This sequence  facilitates the export of the protein into the periplasmatic space.
-+++ TEST TEST TEST +++
 <!-- Add more about the biology of this part here