Difference between revisions of "Part:BBa K404160"

Revision as of 23:52, 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].

Middle Linker ( Gly-Gly-Ser-Gly)x2

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

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

References

Mellon. 2002. Epidermal growth factor receptor and bladder cancer.Postgraduate medical journal78, no. 924 (October): 584-9. doi:10.1136/pmj.78.924.584. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1742539&tool=pmcentrez&rendertype=abstract.
Friedman, Mikaela, Anna Orlova, Eva Johansson, Tove L J Eriksson, Ingmarie Höidén-Guthenberg, Vladimir Tolmachev, Fredrik Y Nilsson, and Stefan Ståhl. 2008. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. Journal of molecular biology376, no. 5: 1388-402. doi:10.1016/j.jmb.2007.12.060. http://www.ncbi.nlm.nih.gov/pubmed/18207161.
Göstring, Lovisa, Ming Tsuey Chew, Anna Orlova, Ingmarie Höidén-guthenberg, Anders Wennborg, Jörgen Carlsson, and Fredrik Y Frejd. 2010. Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects. International Journal of Oncology 36, no. 4 (March): 757-763. doi:10.3892/ijo_00000551. http://www.spandidos-publications.com/ijo/36/4/757.
Hirsch,Fred R, Marileila Varella-Garcia, Paul a Bunn, Michael V Di Maria, Robert Veve, Roy M Bremmes, Anna E Barón, Chan Zeng, and Wilbur a Franklin. 2003. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, no. 20 (October): 3798-807. doi:10.1200/JCO.2003.11.069. http://www.ncbi.nlm.nih.gov/pubmed/12953099.
Nord, K, E Gunneriusson, J Ringdahl, S Ståhl, M Uhlén, and P A Nygren. 1997. Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature biotechnology 15, no. 8 (August): 772-7. doi:10.1038/nbt0897-772. http://www.ncbi.nlm.nih.gov/pubmed/9255793.
Orlova, Anna, Vladimir Tolmachev, Rikard Pehrson, Malin Lindborg, Thuy Tran, Mattias Sandström, Fredrik Y Nilsson, Anders Wennborg, Lars Abrahmsén, and Joachim Feldwisch. 2007. Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer research 67, no. 5 (March): 2178-86. doi:10.1158/0008-5472.CAN-06-2887. http://www.ncbi.nlm.nih.gov/pubmed/17332348.
Walker, R a, and S J Dearing. 1999. Expression of epidermal growth factor receptor mRNA and protein in primary breast carcinomas. Breast cancer research and treatment53, no. 2 (January): 167-76. http://www.ncbi.nlm.nih.gov/pubmed/10326794.
Wikman, M, a-C Steffen, E Gunneriusson, V Tolmachev, G P Adams, J Carlsson, and S Ståhl. 2004. Selection and characterization of HER2/neu-binding affibody ligands. Protein engineering, design & selection : PEDS 17, no. 5 (May): 455-62. doi:10.1093/protein/gzh053. http://www.ncbi.nlm.nih.gov/pubmed/15208403.