Impact
of Nanoparticle Aggregation on Protein Recovery
through a Pentadentate Chelate Ligand on Magnetic Carriers
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Abstract
The
growing need for more efficient separation techniques still
dominates downstream processing of biomolecules, thus encouraging
the continuous development of advanced nanomaterials. In this paper
we present an improved process for recovering recombinant histidine
tagged green fluorescent protein from an <i>E. coli</i> cell
lysate. Superparamagnetic core–shell nanocarriers are functionalized
with a pentadentate chelate affinity ligand and then loaded with metal
ions (Cu<sup>2+</sup>, Ni<sup>2+</sup>, or Zn<sup>2+</sup>). The separation
process yields high binding capacity (250 mg/g), good selectivity,
purity >98%, good recyclability with 90% capacity after 9 cycles,
and long-term stability. We determined the main physical properties
of the magnetite-based nanoparticles such as saturation magnetization
(59 A m<sup>2</sup>/kg), primary particle diameter (22 ± 4 nm),
and specific surface area (89 m<sup>2</sup>/g). Our results show that
this material is a promising tool for bioseparation applications.
One special focus of the work includes analyzing the changes in the
hydrodynamic size distribution using dynamic light scattering and
transmission electron microscopy. We relate these effects to different
interaction levels in the system and discuss how the stronger aggregation
of the magnetite core is the main limiting factor for the separation
yield, leading to a considerable decrease in the number of metal ions
available for biomolecular capture. Otherwise weaker interactions
lead instead to agglomeration effects that have no impact on the binding
capacity of the system. The simple relation between the size of the
aggregated units and the size of the primary particles corresponds
approximately to the relation between the number of existing binding
sites and the actual protein binding in the separation process. Compared
with that, the effect of steric hindrance among proteins is of less
significance