Difference between revisions of "Part:BBa J32015"

Revision as of 12:29, 27 October 2017

PelB leader sequence; directs protein to E. coli periplasmic membrane

The pelB leader sequence is a sequence of amino acids which when attached to a protein, directs the protein to the periplasmic membrane of E. coli, where the sequence is removed by pelB peptidase. It is used to direct coat protein-antigen fusions to the cell surface.

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
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 54
1000
COMPATIBLE WITH RFC[1000]

Improvement

When the 5 aspartate repeated sequence（5D） is added to the back of the pelB，its function will be improved. In order to verify the function of pelB-5D, we constructed piGEM2016-001, piGEM2016-002,piGEM2016-module01* and piGEM2016-module01. piGEM2016-001 and piGEM2016-002 are the expression vectors containing pelB-5D and PETase, the only difference between them is the immanent signal peptide of PETase with piGEM2016-002 was removed.piGEM2016-module01 and piGEM2016-module01* is a vector containing PETase and MHETase, the difference between them is that pelB-5D is added in the front of piGEM2016-module01s'MHETase. SDS-PAGE results of piGEM2016-001 and piGEM2016-002 show that the pelB-5D is working.Further more,SDS-PAGE result of piGEM2016-module01 is obviously better than that of piGEM2016-module01*. In short,All results show that pelB-5D can significantly enhance the secretion of extracellular protein.

Results

To further demonstrate whether the CALB can secrete to the medium by the signal peptide Pelb, the supernatant was directly detected using western blot. CAlB was targeted by His-tag probe. 30ug of protein sample was added in every well. It was obvious that the bands were much more darker in figure B, which indicated the expression of Pelb-CALB#2 was higher than that of Pelb-CALB#1. The predicted molecular weight of fusion protein is different between Pelb-CALB#1 and Pelb-CALB#2, it maybe caused by the changed protein conformation after the codon optimization. Comparing the expression between A and B, it shows that the function of Pelb has been improved remarkablely.

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 https://static.igem.org/mediawiki/2016/0/0b/T--UESTC-China--part21.jpg
-===Improvement by NEFU-China===
-Our team worked to improve the characterization of a signal peptide, BBa_J32015 , part from the iGEM Registry. This part originated from iGEM2006_Duke team can be used as a signal to direct the protein to the periplasmic membrane in E. coli.To improve the function of this part, we made the codon optimization. In order to express and secret the recombinant CALB in E. coli, the 5’-terminal of the gene without its own translational initiation codon was combined with our improved PelB(BBa_K2302005,https://parts.igem.org/Part:BBa_K2302005).
-We demonstrate that expression of the mature portion of the CALB gene fused seamlessly with a pelB signal sequence allows for extracellular production of the CALB enzyme with no extra residues. The mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3). The combination of them resultes in highly successful CALB production. See more details in composite part (BBa_K2302007, BBa_K2302008, BBa_K2302010).The use of the pelB signal sequence for secretory expression is one major reason for the successful production. The pelB signal enabled the translocation of the CALB into the periplasm, where the protein could be correctly folded with the formation of the disulfide bridges.
 ==Results==