Difference between revisions of "Part:BBa K863112"

Revision as of 20:37, 26 September 2012

Cellulose binding domain of C. cellulovorans with T7, RBS, GS-Linker (Freiburg-Standard)

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 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 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