Part:BBa_K3739036
J23100-B0030-Aly01-his-CBM-hutH-B0010
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_ K3739036 to construct the expression system and to express and to purify the protein. Aly01 is a signal peptide from the alginate lyase of V.natriegens, his-tag enable us to purify the linking protein, CBM can bind to cellulose and hutH is a histidine transaminase which converts histidine into urocanic acid.
Biology
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-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_ K3739036 to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_ K3739071 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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 679
Illegal NgoMIV site found at 1115
Illegal NgoMIV site found at 1850
Illegal NgoMIV site found at 2086
Illegal AgeI site found at 137
Illegal AgeI site found at 425
Illegal AgeI site found at 515
Illegal AgeI site found at 752
Illegal AgeI site found at 1579
Illegal AgeI site found at 2075 - 1000COMPATIBLE WITH RFC[1000]
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