Part:BBa_K1932002
SEC2 signal peptides
Sec2 contains a classical SP and a single transmembrane domain. The complete protein appears to be a homologue of a hypothetical secreted protein from B. longum NCC2705 (accession no. NP_695256) and is also encoded by the genomes of B. longum DJO10A (ZP_00121565). A probable acid phosphatase domain (COG0671) was identified in Sec2, which indicates involvement in lipid metabolism.
Characterization:
The prediction of the signal peptide was done with SingalP 4.1 Server (Fig.1), TMpred program (Fig.2) and TMHMM (Fig.3), and the results showed that it could direct the process of transmembrane by cutting the site between amino acid 34 and 35. Since we wanted to fuse it as a domain into our protein, we did the homology modeling and molecular dynamic simulation with the Phyre2 web portal for protein modeling and Hyperchem 8.0 (Fig.4), which showed that the two domains were separate and wouldn’t affect the function of each other.
Fig.1. Analysis of signal peptide on Sec2 by the SignalP 4.1 Server
Fig.2. Analysis of transmembrane-spinning region on Sec2 by the TMpred
Fig.3. Analysis of hydrophobic region on Sec2 by the TMHMM
Fig.4.The simulated structures for Sec2-TAT-Linker-apoptin (Sec2 is highlighted in yellow).
The part of BBa_K1932002 was synthesized and cloned in a pGH vector by Generay Biotechnology. The plasmid was cut by the restriction enzymes, EcoRⅠand PstⅠ, and separated by 1% agarose gel(Fig.5).
Fig.5. (1) Marker; (2) pGH+Sec2 enzyme digestion with EcoR I and Pst I'
The sequence of Sec2 was ligated into the vector pSB1C3 by T4 ligase at 16℃ overnight, and the ligated construct was transformed into the E.coli(Fig.6).
Fig.6. (1) control (only DH5α) ;(2)DH5α that transformed with BBa_K1932002 (the Sec2+pSB1C3 vector)'
To ensure the insertion of the right-size sequence, the sequence of Sec2 was cut again and tested by agarose gel electrophoresis (Fig.7).
Fig.7. (1)Marker; (2) BBa_K1932002 (pSB1C3+Sec2) digested with EcoRI and PstI'
Once the size of this sequence was confirmed, the bacteria containing the construct were sent to the Comate Bioscience Company for DNA sequencing for further verification. The detailed protocols of these experiments were shown in table 1 and table 2.
References:
【1】MacConaill, L. E., Fitzgerald, G. F., & van Sinderen, D. (2003). Investigation of protein export in Bifidobacterium breve UCC2003.Applied and environmental microbiology, 69(12), 6994-7001.
【2】Poquet, I., Ehrlich, S. D., &Gruss, A. (1998). An export-specific reporter designed for gram-positive bacteria: application to Lactococcuslactis. Journal of bacteriology, 180(7), 1904-1912.
【3】Schell, M. A., Karmirantzou, M., Snel, B., Vilanova, D., Berger, B., Pessi, G., ... &Pridmore, R. D. (2002). The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proceedings of the National Academy of Sciences, 99(22), 14422-14427.
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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