Part:BBa_K1932003
TMP1 signal peptides, hypothetical protein
TMP proteins are polypeptides which contain at least one predicted trans-membrane domain. They were identified as active ΔspNuc fusions and were designated Tmp (for putative trans-membrane protein).The fused portion of Tmp1 contains the N-terminal region of a hypothetical protein and is predicted to have a C-out topology, which is typical for transmembrane proteins (Nuc activity of a fusion indicated that the ΔspNuc domain is exported). Proteins similar to Tmp1 have been found only in B. longum NCC2705 (accession no. NP_696268) and DJO10A (ZP_00120937), although no function has been assigned to these proteins.
Characterization:
The signal peptide prediction was done and the results showed that it could direct the process of transferring the membrane by cutting the amino acid between 26 and 27 (Fig.1, Fig.2 and Fig.3).
Fig.1. Analysis of signal peptide on Tmp1 by the SignalP 4.1 Server
Fig.2. Analysis of transmembrane-spinning region on Tmp1 by the TMpred
Fig.3. Analysis of hydrophobic region on Tmp1 by the TMHMM
We did the homology modeling and molecular dynamic simulation (Fig.4), which showed that the two domains were separate and wouldn’t affect the function of each other.
Fig.4. the simulated structures for Tmp1-TAT-Linker-apoptin (Tmp1 is highlighted in yellow).
The part of BBa_K1932003 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+TMP1 enzyme digested with EcoRⅠ and PstⅠ
The sequence of Tmp1 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 transformed with BBa_K1932003 (the TMP1+pSB1C3 vector)
To ensure the insertion of the right-size sequence, the sequence of Tmp1 was cut again and tested by agarose gel electrophoresis (Fig.7).
Fig.7. (1) Marker; (2) BBa_K1932003 (pSB1C3+TMP1) 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. After analyzing the results coming out, the sequence turns out to be correct.
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 tractProceedings 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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