Part:BBa_K929200
N-terminal sortase motif linked to AAV2-VP2 with Myc-tag, kozak and CMV
| CMV_kozak_N-terminal sortase motif_myc-tag_AAV2-VP2 | |
|---|---|
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| BioBrick Nr. | BBa_929200 |
| RFC standard | RFC 25 |
| Requirement | pSB1C3 |
| Source | existing parts: BBa_K404161 |
| Submitted by | [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012] |
The Biobrick Sortase motif linked to AAV2-VP2/3 with myc-tag, kozak and CMV (BBa_K929200) is an altered version of the existing Biobrick pCMV_DARPin-E01_Middle-Linker_[AAV2]-VP23 (BBa_K404161). The DARPin-E01 insertion was replaced by the sortase motif with kozak sequence, Myc-tag and CMV promoter.
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.
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.
Myc-tag
Myc-tag can be used to recognize that the sortase motif is on the surface of the virus
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.
To modify the surface exposed proteins of the recombinant virus we fused the sortase motif to the N-terminus of VP2 cap-gene. VP2 cap-gene is not essential for viral infectivity, what makes it an ideal candidate for the insertions of sequences to retarget the particle. Therefor to reduce and to specify the AAV tropism, the primary receptor motif, heparan sulfat proteoglycan motif, was koncked-out.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2060
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2696
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2619
Illegal SapI site found at 1497
| None |