title: Impact of nanoparticle aggregation on protein recovery through a pentadentate chelate ligand on magnetic carriers.
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 E. coli cell lysate. Superparamagnetic core-shell nanocarriers are functionalized with a pentadentate chelate affinity ligand and then loaded with metal ions (Cu(2+), Ni(2+), or Zn(2+)). 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(2)/kg), primary particle diameter (22 ± 4 nm), and specific surface area (89 m(2)/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.
authors.affiliation: Bioseparation Engineering Group, Mechanical Engineering Faculty, Technische Universität München , Boltzmannstrasse 15, D-85748 Garching, Germany.
authors.forename: P Fraga
authors.initials: PF
authors.lastname: García
authors.forename: M
authors.initials: M
authors.lastname: Freiherr von Roman
authors.forename: S
authors.initials: S
authors.lastname: Reinlein
authors.forename: M
authors.initials: M
authors.lastname: Wolf
authors.forename: S
authors.initials: S
authors.lastname: Berensmeier
mesh_terms: D000327:Adsorption; D001665:Binding Sites; D004492:Edetic Acid / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000302:isolation & purification*; D057446:Hydrodynamics; D008024:Ligands; D060328:Magnetic Phenomena; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008962:Models, Theoretical; D010316:Particle Size; D013696:Temperature; D013997:Time Factors
chemical_list: D008024:Ligands; D058185:Magnetite Nanoparticles; D049452:Green Fluorescent Proteins; D004492:Edetic Acid