Part:BBa_K4825007
JF
"JF is the signal peptide of inulinase originated in Kluyveromyces smarxianu, and utilized in our project's chassis, S.cerevisiae, to target heterologous proteins to the extracellular environment of yeast cells.
This part as a signal peptide can function in S.cerevisiae to enhance the secretion level of heterologous proteins or recombinant enzymes by determining and trafficing the secretion pathway of target genes, but yet the secretion efficiency is not efficient enough.
In our project, this part is used to secrete heterologous protein Cry1518-35 out of S.cerevisiae, of which the efficiency is examined by secreting red fluorescence protein (RFP). This part can be applied as for a more efficient expression of heterologous proteins in S.cerevisiae than the yeast's native signal peptide, especially for the expression of large molecule proteins. "
Usage and Biology
JF, originated from K.smarxianu, is a 74bp signal peptide located in the N-terminal of targeted proteins. Its role in translocating proteins to the periplasmic space or out of the cell starts with the N-region orienting the secretion by interactions with negatively charged phosphate group of the lipid bilayer of the cells; and because of the presence of Lys-Arg at the C-terminal of JF that can be recognized by S.cerevisiae's endoprotease, the translocation ends with the C-region being cleaved by signal peptidase. As a result, the targeted heterologous proteins will be secreted to extracellular space with a much higher level of bioactivity than the bioactivity inside the cells, whereas the signal peptide itself remains in the cytosol.
For continuous secretion expression in modified Saccharomyces cerevisiae, an effective signal peptide is necessary. The different signal peptides, JFm, JF, ScMα were primarily constructed with RFP to compared their functionality. After comparison, Cry was also connected with signal peptides. SDS-page analysis showed that JFm was the most effective one in secretion RFP to supernate. The fluorescence seen in JFm construction and absent in control CEN.Pk2 and intensity quantitatively detected in supernates from different signal peptides construction further verified JFm was the most efficient one. SDS-page analysis for Cry construction also showed that JFm was efficient which was consistent with the previous outcome in RFP construction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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