Part:BBa_K1592000

LIP2 prepro(signal peptide)

13 aa pre/4 XA or XP dipeptides/10 aa pro/KR cleavage site The pre-region of lipase 2 from Yarrowia lipolytica corresponds to the signal sequence. LIP2 prepro consist of 13aa pre-region of lipase2, four XA or XP dipeptides, 10aa pro-region of lipase2, and KR cleavage site. the dipeptides XA and XP are substrates for diamino-peptidase; the dibasic KR cleavage site is substrate for Xpr6p endoproteinase.

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
  COMPATIBLE WITH RFC[1000]

Application of the part

This signal tag, LIP2 prepro, fused to Mcfp3 and constructed a new biobrick (BBa_K1592003), can lead the co-translational translocation of heterologous protein Mcfp-3 to be secreted out of the cell to achieve its function.

Php4d+LIP2 prepro-Mcfp+terminator

We concentrated cell culture solution of test Y.lipolytca JMY1212 cells transformed LIP2 prepro-Mcfp3 plasmid and control wildtype cells, and then separated proteins by SDS-PAGE.

File:HUST-China 2015 results 4.jpg

Figure1: Protein Electrophoresis of LIP2-Mcfp3 (control: the train without plasmid). Mcfp3 protein is about 12kDa.

Figure shows an obvious ~12kDa protein bands of LIP2 prepro-Mcfp3 in test lane, which cannot be found in control lane. This result proves that LIP2 prepro signal peptide can successfully lead the flocculating proteins Mcfp3 into surrounding environment.