Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Potsdam Bioware 2012 iGEM
Date created: 2012-09-23 11:00:00
Date modified: 2015-05-08 01:13:47
N-terminal sortase motif linked to AAV2-VP2 with kozak and CMV
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K929201_sequence (Version 1) |
Description
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 e.g. 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 to allow the ligation by the Sortase. Myc-tag can be used to recognize that the sortase motif is on the surface of the virus.This biobrick should be co-transfected with rAAV-RC (Rep/Cap conatining plasmid) with VP2 knock-out.
CMV
CMV is an immediate-early Cytomegalovirus promoter for high-level expression in many cell-types. The successfull expression of transgene was visible after an introduction of foreign genes in cells with recombinant adenovirus vectors.
Kozak
An additional Kozak sequence upstream of the start codon was inserted to improve the expression of the desired gene.
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.
Capsid
The open reading frame cap on the 3'-end of the AAV-genome encodes the three viral capsid proteins VP1, VP2, and VP3. The transcription of the mRNA is regulated by the p40 promoter. Alternative splicing results in two mRNA the one encodes VP1 and the other VP2/ VP3. Appropriation (Verwendung) of an alternate splice acceptor removes the first AUG start codon for VP1. Thereby mainly formed mRNA encodes the VP2 and VP3 capsid proteins. The first AUG codon in the transcript is the initiation codon for VP3. The translation of VP2 begins at an upstream ACG non-methionin start codon resulting in an approximately 10-fold lower translation of VP2. The stop codon of the three capsid proteins is identical. The stoichiometric ratio of the three capsid proteins VP1, VP2 and VP3 forming the icosahedral form is 1:1:10.
We fused the sortase motif to the N-terminal end of the Cap-gene VP2 because of its presentation on the surface of the virus
Notes
...Source
...| Sequence Annotation | Location | Component / Role(s) |
| CMV+RBS Kozak sequence N-terminal sortase motif AAV2 - VP2/3 Middle linker AgeI | 1,654 662,667 671,685 716,2665 686,709 2666,2671 | promoter feature/promoter feature/rbs ribosome_entry_site CDS feature/cds feature/cds CDS feature/cds CDS feature/binding non_covalent_binding_site |