Cellulose binding Domain of C. Fimi Exoglucanase with T7, RBS, GS-Linker (Freiburg-Standard)
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.
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
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 365
- 1000COMPATIBLE WITH RFC
|family||Cellulose Binding Modul family 2 (pfam00553/cl02709)|
|strain||strain ATCC 484 / DSM 20113 / JCM 1341 / NBRC 15513 / NCIMB 8980 / NCTC 7547|
|type||Cellulose binding domain|