Part:BBa_K2593009

AmyX+6His+LHyal

Biology

AmyX signal peptide (BBa_K2593003): AmyX signal peptide is encoded by a-Amylase gene which is derived from B.amyloliquefaciens. AmyX signal peptide belongs to twin-arginine translocation (Tat) pathway of the Bacillus origin. The Tat pathway has a signature twin-arginine (RR) motif located at the border of the N-terminal domain and the hydrophobic region. The Tat systems of Gram-positive bacteria exhibit interesting differences to those of Gram-negative bacteria, the most striking of which is the absence of a TatB component in virtually all species. In Gram-negative bacteria, membrane-bound TatABC subunits are all essential for activity, whereas Gram-positive bacteria usually contain only TatAC subunits. In Bacillus subtilis, there are two TatAC-type systems, TatAdCd and TatAyCy, operate in parallel with different substrate specificities. in our project AmyX was fused in N-terminal of the LHyal gene for the extracellular export of the expressed protein.
6xHis (BBa_K2593002): Currently His-tags have been extensively applied for recombinant protein expression. Based on previous research, 6His-tag could provide enough spacing for protein folding to prevent crystallization or misfolding of peptide chain, and it is a commonly used in protein purification by chromatography.
LHyal gene(BBa_K2593004) encodes LHAase (Mw=58kD), which is a hyaluronidases from the hyaluronate 3-glycanohydrolases sub-family of leech origin. Hyaluronidase (HAase) is a large family of glycoside hydrolases that predominantly degrades hyaluronic acid (HA), which is a polysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerization.

The usage of this basic part can be seen in our composite part (BBa_K2593006) that contains a promoter and a terminator .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 373
  

Reference

Sunghoon Park, Wolfgang Schumann. Optimization of the Secretion Pathway for Heterologous Proteins in Bacillus subtilis[J].Biotechnology and Bioprocess Engineering,2015:626-627
Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers[J].Scientific Reports, 2014 : 1-2
Jinpeng, Kangzhen, Biosynthesis of hyaluronan oligosaccharides and construction of DNA editing and assembly tools[D]Jiangnan University: Jinpeng,2016.25-27.