Coding

Part:BBa_K929202

Designed by: Potsdam Bioware 2012 iGEM   Group: iGEM12_Potsdam_Bioware   (2012-09-24)

EGF-Receptor_3rd domain with PelB leadersequence, His-tag and C-terminal Sortase motif

PelB, EGF-Receptor_3rd domain, His-tag and C-terminal sortase motif
UP12 BBa K929202-1.png
BioBrick Nr. BBa_K929202
RFC standard RFC 25
Requirement pSB1C3
Source gene synthesis
Submitted by [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]


EGFR 3rd domain.png






















To bring big proteins like EGFR on the surface of the virus, the ligation of two polypeptides in a chemoselective manner could be done. The completely expressed protein EGFR should contain the C-terminal Sortase motif to be recognized by the enzyme Sortase. N-terminal Sortase motif was fused to the N-terminus of VP2/3 gene of adenoassociated Virus in the Biobricks (BBa_K929200) and (BBa_K929201) to allow the ligation by the Sortase.

UP12 Sortase.png


EGF-Receptor

Epidermal growth factor receptor (EGFR), also known as HER1 or ErbB1, is a member of a family of tyrosine kinase receptors which modulates tumor cell differentiation, survival, adhesion, migration and proliferation. EGFR drives tumorigenesis in many cancers such as head and neck cancer, pancreatic cancer, squamous cell lung carcinoma , and colorectal cancer.

EGFR consists of three major functional domains: a cytoplasmic tyrosine kinase domain, a hydrophobic transmembrane domain, and an extracellular ligand-binding domain. In our project we expressed only the extracellular ligand-binding domain, wich binds to the anti EGFR antibody.


PelB leader sequence

The pelB leader sequence was inserted adjacent to the amino-terminus of the sortase motif. PelB leader directs the newly expressed EGFR to the E. coli periplasm where it is cleaved by the membrane-anchored peptidase. Generated desired Protein with N-terminal Sortase motif accumulates in the periplasm and also escape in the extracellular culture media.


Sortase

Sortase is an enzyme which catalyzes specific ligation of two proteins to each other. In the first step the enzym recognizes the C-terminal conserved LPxTG sortase motif and cleaves this motif between Gly and Thr. The obtained thioester intermediat reacts with an N-terminal glycine, regenerating a native amide bond. Gram positive bacteria use the sortase to covalently ligate the proteins to the peptidoglycan layers.


His-Tag

his-tag is used to purify the expressed Protein EGFR in E. coli.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 894
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 664
    Illegal AgeI site found at 1342
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  • Leung K. et al (2012) [http://www.ncbi.nlm.nih.gov/pubmed/22973578 PubMed]
  • Zhixiang W. et al (2012) [http://www.intechopen.com/books/molecular-regulation-of-endocytosis/mutual-regulation-of-receptor-mediated-cell-signalling-and-endocytosis-egf-receptor-system-as-an-exa InTech]
  • Abdullah SE. et al (2012) [http://www.ncbi.nlm.nih.gov/pubmed/22997576 PubMed]
  • Hauser P.S. and Ryen R.O. (2008) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1963442/ PubMed]
[edit]
Categories
Parameters
None