Biology Reference
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.. Addressable Carboxylates from Asp and glu Side Chains
Carboxylates derived from Asp and Glu are in general less favored for
bioconjugation reactions. Compared with Lys, Cys, and Tyr side chains,
carboxylates derived from Asp and Glu are less reactive. Chemical attachment
of amine-containing compounds can be achieved making use of the coupling
reagents EDC and NHS (see Section 4.1.2). An intrinsic problem with this
strategy is that interparticle linkage can occur if the particle itself displays
solvent-exposed amines; such is the case for most VNPs utilized (Table 4.1).
Hydroxylamine can be added to stop the reaction and minimize the formation
of VNP aggregates (hydroxylamines neutralize activated carboxylates)
(Portney
, 2005).
Reactive carboxylates have been identified and utilized on the exterior
surface of CPMV, CCMV,
et al.
Turnip yellow mosaic virus
(TYMV), M13, and
SIRV2 (Bar
et al.
, 2008; Barnhill
et al.
, 2007a,b; Gillitzer
et al.
, 2002;
Steinmetz
, 2008a,b). In addition, reactive carboxylates have been found
on the interior surface of TMV and successful attachment of small chemical
modifiers has been demonstrated (Schlick
et al.
et al.
, 2005) (see also Section 4.3.7
and Table 4.1).
4.3.3 Uilizing Cys Side Chains and the Role of Cys-Added
Mutants
VNPs typically do not display any reactive Cys side chains on the solvent-
exposed exterior capsid surface. In general, if Cys residues are present they
are engaged in the formation of disulfide bonds. Based on the versatility of
thiols for bioconjugation, a variety of Cys-added mutant VNPs have been
generated and have been widely used in a variety of applications (see below).
Naturally occurring reactive Cys side chains have been discovered and
utilized on the solvent-exposed
interior
capsid surface of CPMV (discussed in
Section 4.3.8).
Now, when designing a Cys-added mutant VNP, it is important to
carefully choose the insertion site for the Cys residue. VNPs have a strong
degree of symmetry and polyvalency, and a mutant particle will display
multiple copies of the genetic modification on the particle surface. For
example, a single mutation in a
3 particle will result in 180 modifications
on the intact and assembled virion. Based on the reactivity of the thiol
group of Cys side chains, an undesired effect often observed with Cys-added
mutants is that the particles heavily aggregate via the formation of disulfide
bonds between adjacent particles. Therefore, it is critical to carefully design
Cys-added mutants.
T =
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