Difference between revisions of "Part:BBa K1321340"

Revision as of 23:56, 31 October 2014

Double CBD (dCBD) with N-terminal linker

Double cellulose binding domain (dCBD) using two cellulose binding domains from Trichoderma reesei cellobiohydrolases, with an N-terminal linker and internal linker sequence between the two domains which is derived from the endogenous cellobiohydrolase linker sequence.

Usage and Biology

This part is based on the double cellulose-binding domain construct (CBDcbh2-linker-CBDcbh1) synthesised and characterised by Linder et al [http://dx.doi.org/10.1074/jbc.271.35.21268 (1)] who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.

The two CBDs are from the fungus T. reesei (Hypocrea jecorina) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID [http://www.uniprot.org/uniprot/P62694 P62694]; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID [http://www.uniprot.org/uniprot/P07987 P07987] (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder et al [http://dx.doi.org/10.1074/jbc.271.35.21268 (1)]. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the [http://www.cazy.org/CBM1.html CBM family 1]. The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder et al [http://dx.doi.org/10.1074/jbc.271.35.21268 (1)]; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.

As part of our project we carried out an assay to determine the relative binding ability of CBD-sfGFP fusions to bacterial cellulose.

In our first assay performed to determine the relative strengths of various CBDs’ binding to bacterial cellulose – represented by the percentage fluorescence left from CBDs fused to (sfGFP (RFC25)) bound to bacterial cellulose discs, when subjected to various washes - it was determined that the fusion with dCBD had the greatest binding ability in comparison to four other CBDs fused to sfGFP after three washes with both dH2O and 70% EtOH.

In the same assay, results suggested that dCBD had on average the third greatest ability to bind bacterial cellulose, after three washes with both PBS and 5% BSA.

It was thus decided that dCBD was one of the top three strongest CBDs and was carried forward for use in a metal-binding assay, fusing it to synthetic phytochelatin (insert part number) to show the successful functionalisaton of bacterial cellulose as a metal-removing filter.

Please see below for AutoAnnotator information about the final protein product.

References

1. Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full