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of the particles, were genetically modified with different tags at each end:
a streptavidin-binding peptide was inserted as pIII fusion and a hexa-His
tag as pIX fusion, respectively (see Fig. 4.17, panel B for the location of pIII
and pIX on the M13 virion). The addition of a heterobifunctional linker—a
streptavidin-Ni-NTA complex—to the modified phages resulted in reversible
ring formation of the flexible filamentous particle (Nam
et al.
, 2004) (see also
Chapter 7).
In a similar approach, M13 was modified with a hexa-His tag as a pIX
fusion. At the other end of the phage, selenocysteine (thiol-functional group)
was genetically introduced in pIII; a biotin functionality was subsequently
introduced using thiol-selective chemical coupling. The dual-functionalized
phages were then used as biomolecular tethers, and the elasticity of the
biopolymer was tested using an atomic force microscopy (AFM) approach.
In brief, the phage was immobilized on a surface via the hexa-His tag (pIX);
the other end (pIII) was attached to an AFM tip using a streptavidin-linker
protein (Fig. 4.17) (Khalil
et al.
, 2007a).
4.3.9 General Coupling Protocols, Puriicaion, and Characteri-
zaion Methods
Coupling procedures and reaction conditions vary widely, and optimized
protocols have been developed. The reader is referred to the references given
throughout this chapter and Table 4.1.
Once chemically modified, the functionalized VNP has to be purified
from the reactants and coupling reagents. This is typically achieved using
density gradient ultracentrifugation (Fig. 4.18) followed by ultrapelleting.
With applications in medicine and materials emerging, quality control and
verification of the integrity of the particles after modification are important.
The integrity of VNPs can be verified using a range of methods, such as
electron microscopy, size exclusion chromatography, ion exchange chromato-
graphy, dynamic light scattering, UV/visible spectroscopy, and native and
denaturing gel electrophoresis. It is difficult to generalize techniques used for
testing whether the chemical functionality was successfully introduced and
to quantify the labels per particle. A combination of the above-mentioned
techniques will give a good idea of whether a VNP has been successfully
modified; for example, if modified with a fluorescent molecule, the proteins
can be visualized under UV light in density gradient (Fig. 4.18), and in native
or denaturing gels. UV/visible spectroscopy can be used to quantify the
number of labels. However, additional and more specific techniques may be
chosen for the characterization of the hybrid VNP material, which depend on
the biophysical and biochemical properties of the label itself.
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