3 research outputs found
Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29
We have used isothermal titration calorimetry (ITC) to
identify
and describe binding-coupled equilibria in the interaction between
two protein subunits of archaeal ribonuclease P (RNase P). In all
three domains of life, RNase P is a ribonucleoprotein complex that
is primarily responsible for catalyzing the Mg<sup>2+</sup>-dependent
cleavage of the 5′ leader sequence of precursor tRNAs during
tRNA maturation. In archaea, RNase P has been shown to be composed
of one catalytic RNA and up to five proteins, four of which associate
in the absence of RNA as two functional heterodimers, POP5–RPP30
and RPP21–RPP29. Nuclear magnetic resonance studies of the <i>Pyrococcus furiosus</i> RPP21 and RPP29 proteins in their free
and complexed states provided evidence of significant protein folding
upon binding. ITC experiments were performed over a range of temperatures,
ionic strengths, and pH values, in buffers with varying ionization
potentials, and with a folding-deficient RPP21 point mutant. These
experiments revealed a negative heat capacity change (Δ<i>C</i><sub><i>p</i></sub>), nearly twice that predicted
from surface accessibility calculations, a strong salt dependence
for the interaction, and proton release at neutral pH, but a small
net contribution from these to the excess Δ<i>C</i><sub><i>p</i></sub>. We considered potential contributions
from protein folding and burial of interfacial water molecules based
on structural and spectroscopic data. We conclude that binding-coupled
protein folding is likely responsible for a significant portion of
the excess Δ<i>C</i><sub><i>p</i></sub>.
These findings provide novel structural and thermodynamic insights
into coupled equilibria that allow specificity in macromolecular assemblies
Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions
The
purpose of this work was to elucidate the molecular interactions leading
to monoclonal antibody self-association and precipitation and utilize
biophysical measurements to predict solubility behavior at high protein
concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding
to overlapping epitopes were investigated. Precipitation of mAb-G
solutions was most prominent at high ionic strength conditions and
demonstrated strong dependence on ionic strength, as well as slight
dependence on solution pH. At similar conditions no precipitation
was observed for mAb-R solutions. Intermolecular interactions (interaction
parameter, <i>k</i><sub>D</sub>) related well with high
concentration solubility behavior of both antibodies. Upon increasing
buffer ionic strength, interactions of mAb-R tended to weaken, while
those of mAb-G became more attractive. To investigate the role of
amino acid sequence on precipitation behavior, mutants were designed
by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or
deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation
was observed at high ionic strength for either mutant. The molecular
interactions of mutants were similar in magnitude to those of mAb-R.
The results suggest that presence of hydrophobic groups in the CDR
of mAb-G may be responsible for compromising its solubility at high
ionic strength conditions since deleting these residues mitigated
the solubility issue
RP-HPLC DAR Characterization of Site-Specific Antibody Drug Conjugates Produced in a Cell-Free Expression System
Antibody
drug conjugates (ADCs) harness the target specificity
of a monoclonal antibody (mAb) and the high cytotoxicity of a small
molecule, enabling improved delivery of a potent antitumor agent compared
to traditional chemotherapy for cancer therapy. Only two ADCs have
been marketed, both of which are produced via nonsite-specific conjugation
of the cytotoxic drug to either interchain cysteine (Adcetris) or
lysine (Kadcyla). A growing body of evidence suggests that site-specific
ADCs, because of their payload homogeneity, will improve pharmacokinetics
and have wider therapeutic windows when compared to heterogeneous
ADCs. Previously, we have demonstrated the use of a cell free expression
system (Xpress CF+) for rapid production of site-specific ADCs. Here
we report the generation of a variety of ADCs via conjugation between
a site-specific incorporated non-natural amino acid (nnAA), <i>para</i>-azidomethyl-l-phenylalanine (pAMF), and dibenzocyclooctyl-(polyethylene
glycol)<sub>4</sub> (DBCO-(PEG)<sub>4</sub>) linked payloads using
this platform. We developed a reversed phase HPLC method for drug
to antibody ratio (DAR) characterization, which is applicable to both
reduced and intact ADCs. We demonstrate that these ADCs are of near
complete conjugation and exhibit potent cell killing activity and
in vitro plasma stability. Moreover, we generated an ADC conjugated
at both light and heavy chains, resulting in a DAR close to 4. With
the increased number of payloads, the resultant DAR 4 ADC is potentially
more efficacious than its DAR 2 counterparts, which could further
improve its therapeutic index. These studies have demonstrated the
competency of Xpress CF+ for site-specific ADC production and improved
our understanding of the site-specific ADCs in general