Biomedical subjects

A Chilkoti

Publications and source records attributed to A Chilkoti.

26 records · Page 2Linked to original sources

PubMed1995

The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy.

The interaction forces between biotin and a set of streptavidin site-directed mutants with altered biotin-binding equilibrium and activation thermodynamics have been measured by atomic force microscopy. The AFM technique readily discriminates differences in interaction force between the site-directed (Trp to Phe or Ala) mutants. The interaction force is poorly correlated with both the equilibrium free energy of biotin binding and the activation free energy barrier to dissociation of the biotin-streptavidin complex. The interaction force is generally well correlated with the equilibrium biotin-binding enthalpy as well as the enthalpic activation barrier, but in the one mutant where these two parameters are altered in opposite directions, the interaction force is clearly correlated with the activation enthalpy of dissociation. These results suggest that the AFM force measurements directly probe the enthalpic activation barrier to ligand dissociation.

Bacterial Proteins↗

PubMed1995

Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery.

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetically-engineered streptavidin variants designed for enhanced bioseparation and drug delivery performance. Subunit mixing is accomplished by guanidine thiocyanateinduced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by renaturation and reassociation of mixed tetramers. In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutant. The WT/n49C tetramers can be used for site-specific and stoichiometric attachment of therapeutics/imaging agents or targeting proteins through the genetically-engineered thiol while retaining unhindered access to biotin-binding at the WT subunits. Second, we demonstrate that the His127Cys mutation (H127C) results in a streptavidin mutant that forms a disulfide-linked dimer under non-reducing conditions. Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is controlled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-directed mutant, Trp120Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 10(4) compared to WT streptavidin. The W120 biotin-binding affinity is sufficiently high (Ka approximately equal to 10(7) M-1) to immobilize the mutant on a biotinagarose affinity chromatography column, but the engineered off-rate allows for facile elution with excess biotin at physiological pH, whereas WT streptavidin is irreversibly immobilized on the column. We demonstrate that the purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated targets at the WT subunit, while retaining the reversible separation capabilities of the W120A subunits via biotin-agarose affinity chromatography.

Dimerization↗

PubMed1993

Static secondary ion mass spectrometric investigation of the surface chemistry of organic plasma-deposited films created from oxygen-containing precursors. 3. Multivariate statistical modeling.

Partial least squares (PLS) multivariate statistical models were developed to predict the surface composition and chemistry of a set of model homopolymers based on their static SIMS fragmentation patterns. In the calibration or model-building step, the positive and negative ion static SIMS spectra of different classes of model homopolymers were related to specific chemical attributes of the polymers. The models were then used to examine the surface chemistry of oxygen-containing plasma-deposited films prepared from a variety of precursors. PLS models were developed to predict the surface oxygen concentration and H/C ratios. The results obtained from the PLS models were compared with experimental results.

Analysis of Variance↗

PubMed1993

Analysis of biomedical polymer surfaces: polyurethanes and plasma-deposited thin films.

The surface characterization of biomaterials is important for understanding the biological reactivity of surfaces and for monitoring surface reproducibility and contamination. Electron spectroscopy for chemical analysis (ESCA), secondary ion mass spectrometry (SIMS), contact-angle methods, vibrational spectroscopic methods, and scanning probe microscopies are briefly reviewed. Examples are presented using these methods to characterize RF plasma-deposited surfaces based upon acetone and oxygen for cell culture and Biomer¿ surfaces.

Biocompatible Materials↗

PubMed1991

Static secondary-ion mass spectrometric investigation of the surface structure of organic plasma-deposited films prepared from stable-isotope-labeled precursors. 1. Carbonyl precursors.

Stable-isotope-labeled carbonyl precursors (acetaldehyde, acetone, and 2-butanone) were used to create plasma-deposited films (PDFs), which were then examined by positive- and negative-ion static SIMS. This allowed hydrocarbon (HC) fragments to be distinguished from oxygen-containing fragments in the static SIMS spectra of these PDFs. Both the positive- and negative-ion static SIMS fragmentation patterns of conventional HC and oxygen-containing polymers were qualitatively examined in order to assign structural units on the PDF surface that could account for the sallent features in the static SIMS fragmentation patterns of these PDFs.

Hydrocarbons↗

PubMed1991

Endothelial cell growth on oxygen-containing films deposited by radio-frequency plasmas: the role of surface carbonyl groups.

Polystyrene substrates were modified by radio-frequency plasma deposition from mixtures of various organic vapors (acetone, methane, methanol, and formic acid) and oxygen. The resulting surfaces exhibited a wide range of surface oxygen concentrations, as measured by electron spectroscopy for chemical analysis (ESCA). The surface hydroxyl, carboxyl, and carbonyl groups were derivatized with trifluoroacetic anhydride, trifluoroethanol, or hydrazine, respectively, and their concentrations subsequently determined by ESCA. The growth of bovine aortic endothelial cells was found to increase with the surface carbonyl concentration but did not appear to correlate with the hydroxyl or carboxyl concentrations.

Acetone↗

PubMedDate not supplied

Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein.

A genetically-engineered mutant of cytochrome b5, incorporating a unique cysteine residue, was conjugated to maleimide-terminated oligo(N-isopropylacrylamide). The conjugation of the protein by reaction of the cysteine residue, precisely positioned by site-directed mutagenesis techniques, with an activated oligomer containing only one reactive end group in the oligomer chain permits the site-specific and stoichiometric conjugation of the oligomer with the protein. The protein-oligomer conjugate was shown to exhibit lower critical solution temperature (LCST) behavior, similar to the free oligomer. Furthermore, the LCST behavior of the protein-oligomer conjugate is reversible and allows selective precipitation of the conjugate above its LCST.

Acrylic Resins↗

PubMedDate not supplied

Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin.

Elastin-like polypeptides (ELPs) undergo a reversible, soluble-to-insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (T(t)). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin-ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4-fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of approximately 2 microm form as the temperature is raised to above the T(t). These aggregates persist at all temperatures above the T(t) for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of approximately 12 nm begin to form at the expense of the larger, micron-size aggregates as the temperature is further raised above the T(t). Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron-size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.

Base Sequence↗