Biology Reference
In-Depth Information
the nucleotide sequences of many viruses are known; in addition, infectious
clones have been generated that allow genetic manipulation, cloning, and
expression of a wide range of mutant particles.
A large variety of mutant VNPs have been generated for various
applications; these mutants will be mentioned and discussed in detail
throughout the chapters of this topic. In this section, some of the basic
principles of “smart” mutant design are highlighted. Many virus capsids
have been crystallized and the structure has been solved to near atomic
resolution; with this knowledge in hand, mutants can be designed with
atomic precision. The availability of the structural data allows analyzing
the surface coordinates of the particles for potentially reactive amino acid
side chains. If no such sites are available, amino acids can be introduced by
means of genetics. The site of insertion can be identified using the structure.
It also allows modeling the presentation and orientation of peptides,
proteins, or other ligands that were attached to amino acid side chains by
means of chemistry.
. SeVerAl generAl ClASSeS oF MutAtIonS
1.
Mutations that add novel amino acid side chains to the capsid
surface in order to allow for bioconjugation protocols.
Common
examples are Cys-added mutants. Cys side chains are rarely found on
the solvent-exposed exterior capsid surface; however, the thiol group
of Cys residues is a useful tool for bioconjugation, as the thiol allows
facile coupling to a range of metals or maleimide-activated compounds.
Cys-added mutants have been generated, for example, for MS2, CCMV,
CPMV, and TMV (Klem
et al.
, 2003; Miller
et al.
, 2007; Peabody, 2003;
Wang
, 2002). Lys side chains are also highly versatile groups for
chemical modification and have been introduced into the capsids of
FHV and TMV, both of which do not naturally display any reactive Lys
side chains in their coat protein sequence (Demir & Stockwell, 2002;
Portney
et al.
, 2005).
2. Mutationsthataddnovelaminoacidsidechainsasaffinitytagsto
allow for immobilization or binding with and of other materials.
et al.
Hexa-Histidine tags (that is six constitutive His side chains) are
commonly used as affinity tags for purification of proteins using Ni-NTA
affinity columns. His tags have been introduced into VNPs, and particle
purification over Ni-NTA columns could be carried out (Chatterji
,
2005). His tags have also been used as anchor groups to allow for labeling;
for example, decoration of His-tagged P22 and CPMV with Ni-conjugated
nanogold was shown (Chatterji
et al.
et al.
, 2005; Kang
et al.
, 2008). The Hexa-
Search WWH ::
Custom Search