Difference between revisions of "Part:BBa K863101"
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===Usage and Biology===
 
===Usage and Biology===
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The CBDclos is an N-terminal domain quite close to the start of the protein-sequence (Figure 1). 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 8) 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.
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The CBDcex is an C-terminal domain quite close to the end of the protein-sequence (Figure 1). The NCBI-BLAST identified 100 amino acids (300 bases; green bar) as the protein domain. It belongs to the Cellulose Binding Module family 2 (pfam00553/cl02709) and is part of of the Cellulomonas fimi ATCC 484 exoglucanase gene. In our project the domain was used N-terminal, which made the conserved linking sequence to the glycosyl hydrolase unattractive as a Linker for our fusion-proteins.
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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.
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For the coding sequence 12 bases (4 amino acids) more than the BLAST predicted were taken as conserved sequence upstream and 9 bases (3AS) downstream of the domain to secure natural folding of the domain.
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[[image:Bielefeld2012_Cellubp.jpg|800px|thumb|center|Figure 1: protein-Blast of the Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)]]]
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[[image:Bielefeld2012_Cfimiexo.jpg|800px|thumb|center|Figure 1: protein-Blast of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N ''Cellulomonas fimi'' ATCC 484 exoglucanase gene]]]
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The expressed protein would have 105 amino acids with a molecular weight of 11368.3 and a theoretic pI of 4.56.
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The expressed protein would have 114 amino acids with a molecular weight of 11397.4 and a theoretic pI of 7.85.
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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).
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If the protein concentration is measured by optical density at 280 nm the extinction coefficients would be 27625 M-1 cm-1, assuming all pairs of Cys residues form cystines and 27500 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.
 
It classified the protein as stable.
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<center>
 
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{| class="wikitable"
 
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|Number of amino acids||114
 
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|-
 
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|Molecular weight||11397.4||[Da]
 
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|-
 
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|Theoretical pI||7.85
 
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|-
 
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|Ext. coefficient at 280 nm||27625||[M<sup>-1</sup> cm<sup>-1</sup>] assuming all pairs of Cys residues form cystines
 
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|-
 
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|Ext. coefficient at 280 nm||27500||[M<sup>-1</sup> cm<sup>-1</sup>] assuming all Cys residues are reduced
 
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|}
 
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</center>
 
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Revision as of 12:41, 26 September 2012

Cellulose binding Domain of Cellulomonas Fimi Exoglucanse (Freiburg-Standard)

Cellulose binding domain of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N Cellulomonas fimi ATCC 484 exoglucanase gene] in Standard 25 (Freiburg).


Usage and Biology

The CBDcex is an C-terminal domain quite close to the end of the protein-sequence (Figure 1). The NCBI-BLAST identified 100 amino acids (300 bases; green bar) as the protein domain. It belongs to the Cellulose Binding Module family 2 (pfam00553/cl02709) and is part of of the Cellulomonas fimi ATCC 484 exoglucanase gene. In our project the domain was used N-terminal, which made the conserved linking sequence to the glycosyl hydrolase unattractive as a Linker for our fusion-proteins. For the coding sequence 12 bases (4 amino acids) more than the BLAST predicted were taken as conserved sequence upstream and 9 bases (3AS) downstream of the domain to secure natural folding of the domain.

Figure 1: protein-Blast of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N Cellulomonas fimi ATCC 484 exoglucanase gene]

The expressed protein would have 114 amino acids with a molecular weight of 11397.4 and a theoretic pI of 7.85. If the protein concentration is measured by optical density at 280 nm the extinction coefficients would be 27625 M-1 cm-1, assuming all pairs of Cys residues form cystines and 27500 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 NgoMIV site found at 4
    Illegal AgeI site found at 337
  • 1000
    COMPATIBLE WITH RFC[1000]