Composite
CBDcex(T7)

Part:BBa_K863102

Designed by: Moritz Müller   Group: iGEM12_Bielefeld-Germany   (2012-09-19)

Cellulose binding Domain of C. Fimi Exoglucanase with T7, RBS, GS-Linker (Freiburg-Standard)

Figure 1: Predicted structure of the CBM_3-family made with Cn3D

Cellulose binding domain (CDB) of the Cellulomonas fimi ATCC 484 exoglucanase gene in Standard 25 (Freiburg) under a control of a T7-promoter. This construct was designed to attach proteins of interest via a Standard 25 assembly (Freiburg) to the C-terminal end of the CBD which carries a short linker.


Usage and Biology

The CBDcex is an C-terminal domain at the end of the protein-sequence (Figure 2). 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; Figure 1) 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 2: protein-Blast of the Cellulomonas fimi ATCC 484 exoglucanase gene (the origin of the CBDcex)

The expressed protein would have 112 amino acids with a molecular weight of 11.3 kDa 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 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 365
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//binding/cellulose
//primer/t7
//proteindomain/binding
Parameters
familyCellulose Binding Modul family 2 (pfam00553/cl02709)
functioncellulose binding
originCellulomonas fimi
strainstrain ATCC 484 / DSM 20113 / JCM 1341 / NBRC 15513 / NCIMB 8980 / NCTC 7547
swissproHQ445939
typeCellulose binding domain
uniprotF2XFS7