scholarly journals Pre-clinical Biophysical Characterization of Therapeutic Antibodies in Human Serum by Analytical Ultracentrifugation

2018 ◽  
Vol 114 (3) ◽  
pp. 64a-65a
Author(s):  
Robert T. Wright ◽  
Walter F. Stafford ◽  
Peter J. Sherwood ◽  
David Hayes ◽  
John J. Correia
Keyword(s):  
Human Serum ◽  
Analytical Ultracentrifugation ◽  
Therapeutic Antibodies ◽  
Biophysical Characterization

Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation

2018 ◽  
Vol 550 ◽  
pp. 72-83 ◽  
Author(s):  
Robert T. Wright ◽  
David B. Hayes ◽  
Walter F. Stafford ◽  
Peter J. Sherwood ◽  
John J. Correia
Keyword(s):  
Human Serum ◽  
Analytical Ultracentrifugation ◽  
Serum Proteins ◽  
Therapeutic Antibodies ◽  
Human Serum Proteins

scholarly journals Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 15
Author(s):  
Emilio Reyes-Aldrete ◽  
Erik A. Dill ◽  
Cecile Bussetta ◽  
Michal R. Szymanski ◽  
Geoffrey Diemer ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Analytical Ultracentrifugation ◽  
Atp Hydrolysis ◽  
Size Exclusion ◽  
Radius Of Gyration ◽  
Biophysical Characterization ◽  
X Ray ◽  
Double Stranded Dna ◽  
Symmetric Complex

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

scholarly journals Electrostatics drive oligomerization and aggregation of human Interferon alpha-2a

2021 ◽  
Author(s):  
Christin Pohl ◽  
Marco Polimeni ◽  
Sowmya Indrakumar ◽  
Werner Streicher ◽  
Guenther Peters ◽  
...  
Keyword(s):  
Interferon Alpha ◽  
High Throughput Screening ◽  
Analytical Ultracentrifugation ◽  
Critical Parameters ◽  
Human Interferon ◽  
Biophysical Characterization ◽  
Dipole Interactions ◽  
X Ray Scattering ◽  
Metropolis Monte Carlo

Aggregation and oligomer formation are critical parameters in the field of protein therapeutics and can lead to loss of drug function or even immunogenic responses in patients. Currently two approaches are used to reduce aggregation: (1) finding a suitable formulation which is labor-intensive and requires large protein quantities or (2) engineering the protein by specific, stabilizing mutations, which requires specific knowledge about the protein aggregation pathway. We present a biophysical characterization of the oligomerization and aggregation process by Interferon alpha-2a, a protein drug with antiviral and immunomodulatory properties. We combine high throughput screening with detailed investigations by small-angle X-ray scattering and analytical ultracentrifugation. To get more insight into the molecular mechanism that drives oligomerization and aggregation, we apply molecular Metropolis Monte Carlo simulations. IFNα-2a forms soluble oligomers, which show a fast pH and concentration-dependent equilibrium. We show that attraction between monomers is mainly driven by molecular dipole-dipole interactions, which becomes more pronounced with increasing pH. Repulsion is dominated by ion-ion interaction leading to the formation of insoluble aggregates around the pI which could be prevented by the addition of salt. This study shows how a combination of several methods can help to understand the formation of aggregates and oligomers more systematically and comprehensively, which can lead to better strategies for avoiding aggregation.

scholarly journals Biochemical and Biophysical Characterization of the dsDNA packaging motor from the Lactococcus lactis bacteriophage asccphi28

2020 ◽  
Author(s):  
Emilio Reyes-Aldrete ◽  
Erik A. Dill ◽  
Cecile Bussetta ◽  
Michal R. Szymanski ◽  
Geoffrey Diemer ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Analytical Ultracentrifugation ◽  
Atp Hydrolysis ◽  
Size Exclusion ◽  
Radius Of Gyration ◽  
Biophysical Characterization ◽  
X Ray ◽  
Symmetric Complex ◽  
Exclusion Chromatography

AbstractDouble-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here we describe biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography, analytical ultracentrifugation, small angle x-ray scattering, and negative stain TEM indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other dsDNA phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high resolution structural studies and rigorous biophysical/biochemical analysis.

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