Difference between revisions of "Part:BBa K404160"

 
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<partinfo>BBa_K404160 short</partinfo>
 
<partinfo>BBa_K404160 short</partinfo>
  
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<h2>His-tag</h2>
 +
Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.
 +
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(Smith et al. 1988), (Hoffmann & Roeder 1991)
 +
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. Koerber et al. have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC (Koerber et al. 2007). For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing for an easy insertion into the 453 and/or 587 loop. If the modified capsid bearing a His-tag is being cotransfected with a wild type capsid for the production of mosaic viruses, IMAC helps to not only purify the produced viral particles but also to enrich particles which actually contain the modified proteins. <br>
 +
<h2>CMV</h2>
 +
CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002]. <br>
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<h2>SEG Linker</h2> (BBa_K404300)<br>
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This part is a linker, it can be used to connect two parts and add additional space between them. That can be necessary to avoid interactions between these parts.<br>
 +
<h2>Capsid</h2> (BBa_K404006)<br>
 +
The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).
 +
<html><center><img src="https://static.igem.org/mediawiki/parts/a/a7/Freiburg10_Cap_proteins_VP1_2%263.png" width="600" height="auto"/></center></html><br>
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<h2>ViralBrick 587-KO empty</h2>
 +
(BBa_K4004210)
 +
The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the capsid surface: R484/R487, K532, R585/587. (Trepel et al. 2009). The positively charged arginine residues interact with the HSPGs' negatively charged acid residues. Opie et al. have shown that two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity in AAV2. (Opie et al. 2003). This ViralBrick has been created to introduce this knockout into other constructs. The biobricks with containing this knockout are annotated with „HSPG-ko“.<br>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 23:42, 27 October 2010

pCMV_His-Tag_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-Empty)

His-tag

Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein. The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(Smith et al. 1988), (Hoffmann & Roeder 1991) Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. Koerber et al. have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC (Koerber et al. 2007). For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing for an easy insertion into the 453 and/or 587 loop. If the modified capsid bearing a His-tag is being cotransfected with a wild type capsid for the production of mosaic viruses, IMAC helps to not only purify the produced viral particles but also to enrich particles which actually contain the modified proteins.

CMV

CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

SEG Linker

(BBa_K404300)

This part is a linker, it can be used to connect two parts and add additional space between them. That can be necessary to avoid interactions between these parts.

Capsid

(BBa_K404006)

The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).


ViralBrick 587-KO empty

(BBa_K4004210) The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the capsid surface: R484/R487, K532, R585/587. (Trepel et al. 2009). The positively charged arginine residues interact with the HSPGs' negatively charged acid residues. Opie et al. have shown that two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity in AAV2. (Opie et al. 2003). This ViralBrick has been created to introduce this knockout into other constructs. The biobricks with containing this knockout are annotated with „HSPG-ko“.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 665
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2565
    Illegal SapI site found at 1476