Difference between revisions of "Part:BBa K404250"

Revision as of 02:51, 28 October 2010

pCMV_[AAV2]-VP123ex (ViralBrick-587-Beta-lactamase)
ß-Lactamase motif, inserted into the 453 loop of pCMV_[AAV2]-VP123

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 the phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).

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].

Usage & biology

ß-Lactamase (BLA) is an enzyme produced by some bacteria that cleaves the ß-lactam ring of various antibiotics such as penicillin, rendering them inactive. Because the peptides' N- and C-termini are in close proximity to each other, we created a ViralBrick containing BLA to see if capsid assembly still takes place with such a large insert in place. Assembled capsids can easily be detected by a beta-lactamase assay. Sequence and Features