Part:BBa_K3739040

J23100-B0030-LMT-his-hutH-B0010

LMT is a signal peptide from the lytic murein transglycosylase of V.natriegens, his-tag enable us to purify the linking protein, and hutH is a histidine transaminase which converts histidine into urocanic acid

LMT here represents a signal peptide used to secrect the fusion protein outside the cell. The enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid and his-tag is added to purify the protein. We use BBa_xxxx to construct the expression system and to express and to purify the protein.

Biology

LMT
Lytic murein transglycosylase (LMT) is an enzyme which is able to degrade murein, a component of cell wall of bacteria. There are two kind of LMTs existing in E.coli: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of Vibrio natriegens. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can lead the fused protein secreted out of Vibrio natriegens.
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-LMT-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.
Here, we used BBa_xxxx to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_xxxx and BBa_xxxx.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 661
  Illegal NgoMIV site found at 1097
  Illegal NgoMIV site found at 1832
  Illegal NgoMIV site found at 2068
  Illegal AgeI site found at 407
  Illegal AgeI site found at 497
  Illegal AgeI site found at 734
  Illegal AgeI site found at 1561
  Illegal AgeI site found at 2057
  
• 1000
  COMPATIBLE WITH RFC[1000]