Part:BBa_K3739104
K525998(T7-RBS)-Aly01-his-hutH-B0010
Aly01 here represents a signal peptide used to secrect the fusion protein outside the cell. 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_3739104 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. hutH The HutH comes from Pseudomonas putida. Under natural conditions, many microorganisms can use the histidine ammonia-lyase (HutH) to change L-histidine into trans-urocanate. HutH catalyzes the first step in the degradation of histidine, and the product trans-urocanate 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_K3739006 to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_K3739031 and BBa_K3739107.
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 275
Illegal NgoMIV site found at 711
Illegal NgoMIV site found at 1446
Illegal NgoMIV site found at 1682
Illegal AgeI site found at 111
Illegal AgeI site found at 348
Illegal AgeI site found at 1175
Illegal AgeI site found at 1671 - 1000COMPATIBLE WITH RFC[1000]
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