Difference between revisions of "Part:BBa K863113"

Revision as of 20:47, 26 September 2012

Cellulose binding Domain of C. cellulovorans cellulose binding protein with Reporter GFP

Figure 1: Predicted structure of the CBM_2-family made with Cn3D

Cellulose binding Domain (CBDclos) of Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)] in Standard 25 (Freiburg) under the control of a T7-promoter. This construct was designed as a reporter-fusion-protein to measure the binding capacity of the cellulose binding domain. A GFP is introduced to the C-terminal end of the CBD and connected by a short linker, consisting of four amino acids (Glycine, Serine, Threonine, Glycine).

To that point expression of the protein could not be proven.

Usage and Biology

The CBDclos is an N-terminal domain quite close to the start of the protein-sequence (Figure 2). The NCBI-BLAST identified 92 amino acids (276 bases; green bar) as the protein domain. It belongs to the Cellulose Binding Module family 3 (pfam00942/cl03026; Figure 1) and is part of the very large cellulose binding protein. In which four other carbohydrate binding modules can be found, separated by hydrophobic docking interfaces for cellulase-proteins. This makes the conserved linking sequence unattractive as a Linker for fusion-proteins. For the coding sequence 12 bases (4 amino acids) more than the BLAST predicted were taken as conserved sequence upstream and 6 bases (2AS) downstream of the domain to secure natural folding of the domain.

Figure 2: protein-Blast of the Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)]

The expressed protein would have 105 amino acids with a molecular weight of 11.4 and a theoretic pI of 4.56. If the protein concentration is measured by optical density at 280 nm the extinction coefficients would be 15930 M^-1 cm^-1, assuming all pairs of Cys residues form cystines and 15930 M^-1 cm^-1, assuming all Cys residues are reduced. The [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy Prot-Parameter-tool] predicted the estimated half-life 30 hours in mammalian reticulocytes, (in vitro) more than 20 hours in yeast (in vivo) and more than 10 hours in Escherichia coli (in vivo). It classified the protein as stable. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1067
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 978

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 __NOTOC__
 <partinfo>BBa_K863113 short</partinfo>
+[[Image:Bielefeld2012_pfam00942.jpg|150px|thumb|right|Figure 1: Predicted structure of the CBM_2-family made with Cn3D]]
+Cellulose binding Domain (CBDclos) of ''Clostridium cellulovorans'' [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)] in Standard 25 (Freiburg) under the control of a T7-promoter. This construct was designed as a reporter-fusion-protein to measure the binding capacity of the cellulose binding domain. A GFP is introduced to the C-terminal end of the CBD and connected by a short linker, consisting of four amino acids (Glycine, Serine, Threonine, Glycine).
-Cellulose binding Domain from Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene] with T7, RBS linked with two amino acids (GS) to a GFP with His-tag.
+To that point expression of the protein could not be proven.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+The CBDclos is an N-terminal domain quite close to the start of the protein-sequence (Figure 2). The NCBI-BLAST identified 92 amino acids (276 bases; green bar) as the protein domain. It belongs to the Cellulose Binding Module family 3 (pfam00942/cl03026; Figure 1) and is part of the very large cellulose binding protein. In which four other carbohydrate binding modules can be found, separated by hydrophobic docking interfaces for cellulase-proteins. This makes the conserved linking sequence unattractive as a Linker for fusion-proteins.
+For the coding sequence 12 bases (4 amino acids) more than the BLAST predicted were taken as conserved sequence upstream and 6 bases (2AS) downstream of the domain to secure natural folding of the domain.
+[[image:Bielefeld2012_Cellubp.jpg|800px|thumb|center|Figure 2: protein-Blast of the Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)]]]
+The expressed protein would have 105 amino acids with a molecular weight of 11.4 and a theoretic pI of 4.56.
+If the protein concentration is measured by optical density at 280 nm the extinction coefficients would be 15930 M<sup>-1</sup> cm<sup>-1</sup>, assuming all pairs of Cys residues form cystines and 15930 M<sup>-1</sup> cm<sup>-1</sup>, assuming all Cys residues are reduced. The [http://web.expasy.org/cgi-bin/protparam/protparam ExPASy Prot-Parameter-tool] predicted the estimated half-life 30 hours  in mammalian reticulocytes, (in vitro) more than 20 hours in yeast  (in vivo) and more than 10 hours in ''Escherichia coli'' (in vivo).
+It classified the protein as stable.
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>