Difference between revisions of "Part:BBa K258008"

(→‎Export of recombinant proteins in Escherichia coli using ABC transporter of Erwinia chrysanthemi)
 
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== Export of recombinant proteins in Escherichia coli using ABC transporter of Erwinia chrysanthemi  ==
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<partinfo>BBa_K258008 short</partinfo>
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EGF-TliA, EGF-LARD1 fusion proteins were secreted when ABC transporter was present. E. coli harboring the PrtDEF of E. chrysanthemi secreted more fusion proteins than without ABC transporter. In fact, in enzyme activation assay(with Hitachi Spectrophotometer) we observed that although our protein -EGF- has disulfide bonds, it was excreted succesfully. In addition, when compearing with lack of ABC domain, we saw that protein excretion into medium (supernatant) is 10-fold more with ABC transporter-PrtDEF-. PrtDEF functioned well at 37°C, the optimum growth temperature for E. coli
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<br>Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases
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secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore
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the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF
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genes) in Escherichia coli and tested the ability of this ABC transporter to export proteins carrying
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C-terminal fragments LARD1 or whole TliA (the longest LARD) of
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Pseudomonas fluorescens SIK W1.
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 +
<br>EGF-TliA, EGF-LARD1 fusion proteins were secreted when ABC transporter was present. E. coli harboring the PrtDEF of E. chrysanthemi secreted more fusion proteins than without ABC transporter. In fact, in enzyme activation assay(with Hitachi Spectrophotometer) we observed that although our protein -EGF- has disulfide bonds, it was excreted succesfully. In addition, when compearing with lack of ABC domain, we saw that protein excretion into medium (supernatant) is 10-fold more with ABC transporter-PrtDEF-. PrtDEF functioned well at 37°C, the optimum growth temperature for E. coli.
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In our project Wound Dressing, for excretion of EGF and KGF into supernatant,we used the plasmid.It is a device which contains three genes-prtD,prtE and prtF- in order to form ATP Binding Cassette(ABC) transporter domain on E.coli membrane.Thus,the plasmid is not used for digestion or ligation.The plasmid works with constutively on "tetR" promoter.
  
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== Sequence and Features ==
 
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<br>[1]PrtDEF: ABC transporter of Erwinia chrysanthemi for PrtA  
 
<br>[1]PrtDEF: ABC transporter of Erwinia chrysanthemi for PrtA  
 
<br>[2]EGF: Human Epidermal Growth Factor
 
<br>[2]EGF: Human Epidermal Growth Factor
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<html>
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<div align="center" style="padding-left: 66px; padding-top: 8px;"><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2642768/"><img style="border: 0px solid ; width: 447px; height: 286px;" alt="w6" src="https://static.igem.org/mediawiki/2009/1/1f/PrtDEF.jpg"></a></div></html>
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'''Figure''':Proposed model to explain blockage of secretion for proteins containing disulfide bonds. (A) The hybrid carrying a preformed disulfide
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bond approaches the channel entrance, is recognized by the secretion machinery (PrtD), but cannot go across the channel. (B) Proteins are
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stacked inside the channel during secretion. Disulfide bonds may be formed at this stage. (C)Inclusion body formations of protein can not enter into the domain nor pass through the domain.
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== Sequence and Features ==
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<partinfo>BBa_K258008 SequenceAndFeatures</partinfo>

Latest revision as of 07:30, 18 October 2009

Export of recombinant proteins in Escherichia coli using ABC transporter of Erwinia chrysanthemi


Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export proteins carrying C-terminal fragments LARD1 or whole TliA (the longest LARD) of Pseudomonas fluorescens SIK W1.


EGF-TliA, EGF-LARD1 fusion proteins were secreted when ABC transporter was present. E. coli harboring the PrtDEF of E. chrysanthemi secreted more fusion proteins than without ABC transporter. In fact, in enzyme activation assay(with Hitachi Spectrophotometer) we observed that although our protein -EGF- has disulfide bonds, it was excreted succesfully. In addition, when compearing with lack of ABC domain, we saw that protein excretion into medium (supernatant) is 10-fold more with ABC transporter-PrtDEF-. PrtDEF functioned well at 37°C, the optimum growth temperature for E. coli. In our project Wound Dressing, for excretion of EGF and KGF into supernatant,we used the plasmid.It is a device which contains three genes-prtD,prtE and prtF- in order to form ATP Binding Cassette(ABC) transporter domain on E.coli membrane.Thus,the plasmid is not used for digestion or ligation.The plasmid works with constutively on "tetR" promoter.


[1]PrtDEF: ABC transporter of Erwinia chrysanthemi for PrtA
[2]EGF: Human Epidermal Growth Factor

w6

Figure:Proposed model to explain blockage of secretion for proteins containing disulfide bonds. (A) The hybrid carrying a preformed disulfide bond approaches the channel entrance, is recognized by the secretion machinery (PrtD), but cannot go across the channel. (B) Proteins are stacked inside the channel during secretion. Disulfide bonds may be formed at this stage. (C)Inclusion body formations of protein can not enter into the domain nor pass through the domain.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 210
    Illegal PstI site found at 793
    Illegal PstI site found at 1140
    Illegal PstI site found at 3055
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4344
    Illegal PstI site found at 210
    Illegal PstI site found at 793
    Illegal PstI site found at 1140
    Illegal PstI site found at 3055
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1259
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 210
    Illegal PstI site found at 793
    Illegal PstI site found at 1140
    Illegal PstI site found at 3055
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 210
    Illegal PstI site found at 793
    Illegal PstI site found at 1140
    Illegal PstI site found at 3055
    Illegal NgoMIV site found at 4706
    Illegal AgeI site found at 3189
  • 1000
    COMPATIBLE WITH RFC[1000]