Part:BBa_K3739031
J23100-B0030-his-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_K3739031 to construct the expression system and to express and to purify the protein.
Biology
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-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. We used BBa_ K3739031 to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_K3739104 and BBa_K3739107.
Characterization
1. Agarose Gel Electrophoresis When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target (1877bp).
Fig.1 The result of colony PCR. Plasmid pET-28a (+).
References
1. 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 232
Illegal NgoMIV site found at 668
Illegal NgoMIV site found at 1403
Illegal NgoMIV site found at 1639
Illegal AgeI site found at 305
Illegal AgeI site found at 1132
Illegal AgeI site found at 1628 - 1000COMPATIBLE WITH RFC[1000]
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