Part:BBa_K3739106
K525998(T7-RBS)-Aly01-his-CBM-hutH-B0010
Aly01 here represents a signal peptide used to secrect the fusion protein outside the cell, his-tag enable us to purify the linking protein. The fusion protein CBM-hutH works on the surface of single cell Phaeocystis. globosa and the enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid. His-tag is added to purify the protein. We use BBa_K3739106 to construct the expression system and to express and to purify the protein.
Biology
Aly01
Aly01 is alginate lyase from Vibrio natriegens SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in E. coli. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently.
CBM
Cellulose enzymes have two domains, and the one that helps bind to cellulose is called cellulose binding module (CBM), and therefore it helps our fusion protein bind to cellulose-rich cell wall. Here we choose the CBM of CenA from Cellulomonas fimi, which has been successfully expressed in Escherichia coli.
hutH
The HutH comes from Pseudomonas putida. Under natural conditions, many microorganisms can use the histidine ammonia-lyase (HutH) to change L-histidine into urocanic acid. HutH catalyzes the first step in the degradation of histidine, and the product urocanic acid is further metabolized to glutamate. This enzyme could be found in the liver of vertebrates and in bacteria such as Escherichia coli, Salmonella and Pseudomonas. It is specific for L-histidine and can be inhibited by D-histidine or imidazole. The active center of the enzyme is thought to be dehydroalanine.
Usage
In order to easily purify HutH, we added a his-tag (6*His) at the C-terminal. We ligased the strong promoter (BBa_J23100) and the parts (RBS-Aly01-his-CBM-hutH-Terminator) on the expression vector pET-28a (+) by standard assembly. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing. Here, we used BBa_K3739106 to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_K3739109 and BBa_xxxx.
References
1.https://parts.igem.org/Part:BBa_K3185005
2.Meng, Q. et al. Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase. Journal of the science of food and agriculture 101, 5154-5162, doi:10.1002/jsfa.11161 (2021).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 653
Illegal NgoMIV site found at 1089
Illegal NgoMIV site found at 1824
Illegal NgoMIV site found at 2060
Illegal AgeI site found at 111
Illegal AgeI site found at 399
Illegal AgeI site found at 489
Illegal AgeI site found at 726
Illegal AgeI site found at 1553
Illegal AgeI site found at 2049 - 1000COMPATIBLE WITH RFC[1000]
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