Difference between revisions of "Part:BBa K346029:Design"

 
 
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===Design Notes===
 
===Design Notes===
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...
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'''Background'''
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Lpp-OmpA(46-159) fusions have proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase[2], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[3] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on the outer membrane protein A (OmpA) suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[2].
 +
 
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In addition to the assembly part which is played by OmpA, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The amino termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity[1]. To conclude, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display.
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 +
'''Designation:'''
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The design is like what we have done to that for mercury binding.
 +
 
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[[Image:surface display.jpg]]
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 +
In our design, we construct a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide(MBP) for lead. the signal peptide of the N-termini of this fusion protein targets the protein on the membrane while the trans membrane domain of Ompa serves as an anchor. MBP is on the externally exposed loops of OmpA, which is anchored to the outer membrane. The fusion protein gene lpp-ompA-mbp is driven by the T7 promoter.
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===Source===
 
===Source===
  
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...
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plasmid PSD-MBD from Anne O. Summers
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plasmid containing PbrR from Pro. Chuan He
  
 
===References===
 
===References===
 +
 +
[1]Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.
 +
 +
[2]Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.
 +
 +
[3]Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. ProcNatl Acad Sci U S A 90, 10444–10448
 +
 +
[4]Daugherty, P. S., Olsen, M. J., Iverson, B. L. & Georgiou, G. (1999).Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng 12, 613–621.
 +
 +
[5]Song, L., Caguiat, J., Li, Z., Shokes, J., Scott, R. A., Olliff, L. &Summers, A. O. (2004). Engineered single-chain, antiparallel,coiled coil mimics the MerR metal binding site. J Bacteriol 186,1861–1868.
 +
 +
[6]Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719

Latest revision as of 04:15, 26 October 2010

T7p(BBa_I719005)+RBS(B0034)+Lpp-OmpA+MBP(surface display of lead binding peptide)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Background

Lpp-OmpA(46-159) fusions have proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase[2], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[3] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on the outer membrane protein A (OmpA) suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[2].

In addition to the assembly part which is played by OmpA, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The amino termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity[1]. To conclude, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display.

Designation:

The design is like what we have done to that for mercury binding.

Surface display.jpg

In our design, we construct a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide(MBP) for lead. the signal peptide of the N-termini of this fusion protein targets the protein on the membrane while the trans membrane domain of Ompa serves as an anchor. MBP is on the externally exposed loops of OmpA, which is anchored to the outer membrane. The fusion protein gene lpp-ompA-mbp is driven by the T7 promoter.




Source

plasmid PSD-MBD from Anne O. Summers

plasmid containing PbrR from Pro. Chuan He

References

[1]Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.

[2]Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.

[3]Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. ProcNatl Acad Sci U S A 90, 10444–10448

[4]Daugherty, P. S., Olsen, M. J., Iverson, B. L. & Georgiou, G. (1999).Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng 12, 613–621.

[5]Song, L., Caguiat, J., Li, Z., Shokes, J., Scott, R. A., Olliff, L. &Summers, A. O. (2004). Engineered single-chain, antiparallel,coiled coil mimics the MerR metal binding site. J Bacteriol 186,1861–1868.

[6]Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719