Part:BBa_K404201

ViralBrick-453-BAP
The Biotinylation Acceptor peptide motif, ready for insertion into the 453 loop

ViralBrick-453-BAP
BioBrick Nr.	BBa_K404201
RFC standard	RFC 25RFC 10
Requirement	backbone without SspI, SalI, BamHI and PvuII (e.g. pSB1C3_VCK)
Source	synthetic
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

All capsid coding parts (e.g. (AAV2)-RepVP123) of the Virus Construction Kit designed by the iGEM Team Freiburg contain single cutting restriction sites on the side of the sequences coding for the two major surface exposed loops.
Using these restriction sites it is possible to insert functional motifs into the coding sequence for the viral capsid.

This BioBrick contains one of these functional motifs fanked by bilateral linkers and the viral sequences that code for the loop.
This category of BioBricks was termed ViralBrick to clarify that cloning does not function with the usual iGEM RFC restriction sites but with the mentioned ViralBrick restriction sites.

The four restriction sites for the ViralBrick insertion were designed in a way that the amino acid sequence of the viral capsid could be absolutely conserved. This was advisable because even slight changes in the viral capsid lead to drastically changed interactions with cellular surface receptors. For this reason we decided to insert these restriction sites in stead of using BioBrick assembly that would define at least two amino acids in the viral loop.

Schematic representation of the BioBrick with all relevant annotations

Restriction sites

Restriction site	Enzyme	Recognition sequence	Activity	Temp.
upstream 453	[http://www.neb.com/nebecomm/products/productR3132.asp SspI-HF]		25;100;0;100	37°C
downstream 453	[http://www.neb.com/nebecomm/products/productR3138.asp SalI-HF]		10;100;100;100	37°C

Modification of the Viral Capsid of the AAV2 using Viral Bricks

Three dimensional representation of the AAV2 showing the amino acids of the 453 and 587 loops that are coded by the corresponding Viral Bricks

For therapeutical applications in human gene transfer the broad tropism for heparan sulfate proteoglycan (HSPG) has to be knocked-out and a novel tropism has to be inserted.

This retargeting can be realized either by insertion of functional motifs into the two major surface exposed loops or by fusion of these motifs to the N-terminus of the viral coat proteins.

The graphic on the right shows parts of the three-dimensional structure of a viral coat protein. The parts of the loop regions that are coded in the ViralBricks are shown in purple for the 453 loop and in blue for the 587 loop.

Cloning of Viral Bricks into capsid coding parts

In order to make loop insertions more convenient the following restriction sites were inserted into all capsid coding parts and already existing restriction sites were removed from the constructs.

The choice of these restriction sites was reasoned by enzyme performance, buffer compabilities and the number of existing restriction sites that had to be removed at other positions.

All restriction endonucleases were purchased from NEB.

Restriction site	Enzyme	Recognition sequence	Activity	Temp.
upstream 453	[http://www.neb.com/nebecomm/products/productR3132.asp SspI-HF]		25;100;0;100	37°C
downstream 453	[http://www.neb.com/nebecomm/products/productR3138.asp SalI-HF]		10;100;100;100	37°C
upstream 587	[http://www.neb.com/nebecomm/products/productR3136.asp BamHI-HF]		100;50;10;100	37°C
downstream 587	[http://www.neb.com/nebecomm/products/productR3151.asp PvuII-HF]		0;25;0;100	37°C

Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide

The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA).
The purified BirA biotin ligase that was kindly provided from Avidity.com

References:

IMAC purification via Viral Brick: The Histidin Affinity 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.

References:

Targetingpeptides via ViralBrick: The RGD Motif

References:

Coupling Antibodies to the Viral Surface via ViralBrick: The Z34C Motif

The idea of this targeting approach is to utilise a minimized fragment of the Staphylococcal Protein A that was first described in Staphylococcus aureus. This gram-positive bacteria has evolved the 508 amino acid long protein A that has a high affinity for the Fc-domain of antibodies to protect itself from the immune systeme. Binding to the constant region of the antibodies is accomplished by the Z-Domain of Protein A that is 58-59 amino acids long, has alone a high affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle structure. In [Braisted & Wells; 1996] the authors reduced the secundary structure to an two-helix bundle. This size reduction has lead to an drastic reduction of the affinity for IgG (>10^5 fold) which could be recovered by 13 amino acid exchanges resulting in a 38 amino acid long peptide with an satisfying affinity for IgG (Kd = 185 nM) termed Z38. This binding domain was subsequently improoved in [Starovasnik et al.; 1997] by the insertion of a disulfide bridge connecting the ends of the helices leading to the binding domain Z34C which shows an increased affinity for IgG (Kd = 20 nM).
This engineered antibody binding domain of 34 amino acids was then inserted into capsids of different viral vectors amongst others also the AAV. In [Ried et al.; 2002] the Z34C domain was inserted at position 587 into the capsid of the AAV resulting in viral vector that can be targeted to different target cells without genetic engineering. This targeting approach was then improved in [Gigout et al.; 2005] by the creation of mosaic vectors that contain only ~25% of recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude more infectiosity compared to all-mutant viruses.

References:

[Braisted & Wells; 1996]

[Starovasnik et al.; 1997]

[Ried et al.; 2002]

[Gigout et al.; 2005]

Risk assessment of the Adeno-associated Virus

Viral Systems based on the Adeno-associated Virus of the serotype 2 are to handle under Biosafety level 1 according to the german Act on Genetic Engineering the specification of the ZKBS on working with the AAV. In the United States, the legal regulations are comparable according to the National Institutes of Health (NIH) which classifies the AVV-2 as BSL-1 in the Appendix B. This general classification is restricted to experiments that use the ITRs (Inverted Terminal Repeats) of the AAV2 and do not contain hazzardous gene sequences on the vector plasmid.
Please consider special provisions of law in your contry before using parts that contribute the production of genetically modified viral vectors.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]