Use of Dodecahedron “VLPs” as an Alternative to the Whole Adenovirus - Adenovirus: Methods and Protocols - page 61

when the penton base and fi ber are overexpressed in the Baculovirus
expression system, a symmetrical dodecameric structure mimicking
the overall “collapsed” virion is obtained [ 2 ]. A fi berless dodecahe-
dron called Base-Dodecahedron (Bs-Dd) can be also produced [ 3 , 4 ],
indicating that the dodecamerization information is located in the
penton base of some but not all adenovirus serotypes (e.g., Ad3
and Ad7). The production and purifi cation of these unconven-
tional particles can be achieved in a few steps to provide a relatively
high concentration (3 mg/mL) which corresponds to two to three
orders of magnitude more than the adenovirus titer. Both Pt-Dd
and Bs-Dd are effi ciently internalized by cells, making them good
tools for versatile applications in both fundamental research like
receptor fi shing, traffi cking studies [ 5 - 7 ], and in vectorology such
as DNA and protein delivery or vaccinology [ 8 - 11 ]. Both, ease of
production and safety properties of these genome-devoid particles,
in combination with the good structural and functional knowl-
edge, led to increased interest around these VLPs in the past
5 years [ 7 , 12 , 13 ]. In this chapter, state of the art protocols around
adenovirus type 3 dodecahedron are described.

2 Materials

2.1 Production:
Cell Culture and
Baculovirus Infection

1. High Five cell (Invitrogen).
2. Express Five Serum-Free Medium (Invitrogen).
3. Rotative shaker Certomat (Sartorius).
4. Thermostated incubator.
5. Laminar Flow Sterile Hood.

2.2 Purifi cation
and Dialysis

1. Ultracentrifuge.
2. SW41 rotor and bucket set (Beckman).
3. Ultraclear 12 mL ultracentrifuge tubes.
4. HBS-G solution: 20 mM HEPES pH 7.4, 150 mM NaCl,
10 % glycerol ( see Note 1 ).
5. 50 % Sucrose in HBS-G solution.
6. Dialysis Tube with MWCO 100 kDa.

1. Protein in amine-free buffer: PBS, HEPES…no TRIS! ( see
Note 2 ).
2. Hoechst 33258 (Sigma).
3. Fluorescent dyes: Alexa Fluor 488 or 555 (Invitrogen) or Cy5
mono-NHS: (GE-Healthcare).
4. Biotin EZ-Sulfo-NHS (Thermo Scientifi c).
5. Dialysis Tube with MWCO 100 kDa ( see Note 3 ).

2.3 Dodecahedron
Labeling and Cellular
Traffi cking