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 Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms

2018 ◽  
Vol 22 (2) ◽  
pp. 57
Author(s):  
Ratih Asmana Ningrum ◽  
Widdya Kusuma Wardhani ◽  
Ike Wahyuni ◽  
Apon Zaenal Mustopa
Keyword(s):  
Interferon Alpha ◽  
Protein Characterization ◽  
Protein Yield ◽  
Size Exclusion ◽  
Human Interferon ◽  
Two Dimensional ◽  
Cancer Treatments ◽  
Sds Page ◽  
Bca Assay

     Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.

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

scholarly journals Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

2015 ◽  
Vol 71 (4) ◽  
pp. 986-995 ◽  
Author(s):  
C. M. D. Swarbrick ◽  
M. A. Perugini ◽  
N. Cowieson ◽  
J. K. Forwood
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Functional Characterization ◽  
Fatty Acyl ◽  
Size Exclusion ◽  
X Ray Scattering ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Enzyme Families

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on theEscherichia colistructure.Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of theY. pestisTesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

scholarly journals Engineering and characterization of a recombinant human interferon alpha-2b fused with apolipoprotein A-I possessing prolonged action

2020 ◽  
Author(s):  
Maria Pykhtina ◽  
Vladimir Romanov ◽  
Anastasiya Kotlyarova ◽  
Evgeniy Demidov ◽  
Svetlana Bannikova ◽  
...  
Keyword(s):  
Interferon Alpha ◽  
Human Interferon ◽  
Prolonged Action ◽  
Interferon Alpha 2B ◽  
Apolipoprotein A

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.

Identification and characterization of human interferon alpha inhibitors through a WISH cell line-based reporter gene assay

2020 ◽  
Vol 94 ◽  
pp. 103372
Author(s):  
Milagros Bürgi ◽  
Paola Hernández ◽  
Mauricio Cabrera ◽  
Hugo Cerecetto ◽  
Mercedes González ◽  
...  
Keyword(s):  
Cell Line ◽  
Reporter Gene ◽  
Interferon Alpha ◽  
Reporter Gene Assay ◽  
Human Interferon ◽  
Gene Assay ◽  
Identification And Characterization

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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