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A Chilkoti

Publications and source records attributed to A Chilkoti.

At least 19 recordsLinked to original sources
PubMed2004

Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.

Amino Acid Sequence↗
PubMed2001

Enhanced uptake of a thermally responsive polypeptide by tumor cells in response to its hyperthermia-mediated phase transition.

Elastin-like polypeptides (ELPs) composed of a VPGXG repeat undergo a reversible phase transition in aqueous solution. They are hydrophilic and soluble in aqueous solution below their transition temperature (T(t)), but they become hydrophobic and aggregate when the temperature is raised above their T(t). In this study, we examine the quantitative uptake of a fluorescence-labeled, thermally responsive ELP as a function of ELP concentration between 5 and 15 microM in solution in response to hyperthermia by three cultured cancer cell lines. Flow cytometry of fluorescein-ELP conjugates showed that hyperthermia enhanced the cellular uptake of the thermally responsive ELP in human ovarian carcinoma cells (SKOV-3) and in HeLa cells at a concentration of 10 microM or higher, but not at a concentration of 5 microM, as compared with the uptake of a thermally inactive ELP control. In FaDu cells, hyperthermia stimulated uptake of the thermally responsive ELP at all solution concentrations of ELP between 5 and 15 microM. In particular, a >2-fold greater uptake of thermally responsive ELP compared with the thermally inactive control ELP was observed for FaDu cells at a solution concentration of 15 microM in heated cells. Confocal fluorescence microscopy of tumor cells incubated with a rhodamine conjugate of the thermally responsive ELP showed that the cytoplasm was uniformly stained below the T(t). Above the T(t), fluorescent particles were observed in the cytoplasm, suggesting that these particles are aggregates of the thermally responsive polypeptide resulting from the ELP phase transition. These studies demonstrate that the endocytotic uptake of a thermally responsive ELP is significantly enhanced by the thermally triggered phase transition of the polypeptide.

Carcinoma, Squamous Cell↗
PubMed2001

Interfacial phase transition of an environmentally responsive elastin biopolymer adsorbed on functionalized gold nanoparticles studied by colloidal surface plasmon resonance.

The change in optical properties of colloidal gold upon aggregation has been used to develop an experimentally convenient colorimetric method to study the interfacial phase transition of an elastin-like polypeptide (ELP), a thermally responsive biopolymer. Gold nanoparticles, functionalized with a self-assembled monolayer (SAM) of mercaptoundecanoic acid onto which an ELP was adsorbed, exhibit a characteristic red color due to the surface plasmon resonance (SPR) of individual colloids. Raising the solution temperature from 10 degrees C to 40 degrees C thermally triggered the hydrophilic-to-hydrophobic phase transition of the adsorbed ELP resulting in formation of large aggregates due to interparticle hydrophobic interaction. Formation of large aggregates caused a change in color of the colloidal suspension from red to violet due to coupling of surface plasmons in aggregated colloids. The surface phase transition of the ELP was reversible, as seen from the reversible change in color upon cooling the suspension to 10 degrees C. The formation of colloidal aggregates due to the interfacial phase transition of adsorbed ELP was independently verified by dynamic light scattering of ELP-modified gold colloids as a function of temperature. Colloidal SPR provides a simple and convenient colorimetric method to study the influence of the solution environment, interfacial properties, and grafting method on the transition properties of ELPs and other environmentally responsive polymers at the solid-water interface.

Adsorption↗
PubMed2001

Drug targeting using thermally responsive polymers and local hyperthermia.

We report a new thermal targeting method in which a thermally responsive drug carrier selectively accumulates in a solid tumor that is maintained above physiological temperature by externally applied, focused hyperthermia. We synthesized two thermally responsive polymers that were designed to exhibit a lower critical solution temperature (LCST) transition slightly above physiological temperature: (1) a genetically engineered elastin-like polypeptide (ELP) and (2) a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). The delivery of systemically injected polymer-rhodamine conjugates to solid tumors was investigated by in vivo fluorescence video microscopy of ovarian tumors implanted in dorsal skin fold window chambers in nude mice, with and without local hyperthermia. When tumors were heated to 42 degrees C, the accumulation of a thermally responsive ELP with a LCST of 40 degrees C was approximately twofold greater than the concentration of the same polymer in tumors that were not heated. Similar results were also obtained for a thermally responsive poly(NIPAAM-co-AAm), though the enhanced accumulation of this carrier in heated tumors was lower than that observed for the thermally responsive ELP. These results suggest that enhanced delivery of drugs to solid tumors can be achieved by conjugation to thermally responsive polymers combined with local heating of tumors.

Acrylamides↗
PubMed2001

Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia.

Elastin-like polypeptides (ELPs) are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. They are soluble in aqueous solutions below their transition temperature (T1) but hydrophobically collapse and aggregate at temperatures greater than T1. We hypothesized that ELPs conjugated to drugs would enable thermally targeted drug delivery to solid tumors if their T1 were between body temperature and the temperature in a locally heated region. To test this hypothesis, we synthesized a thermally responsive ELP with a T1 of 41 degrees C and a thermally unresponsive control ELP in Escherichia coli using recombinant DNA techniques. In vivo fluorescence videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors (SKOV-3 ovarian carcinoma and D-54MG glioma) implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately 2-fold increase in tumor localization compared to the same polypeptide without hyperthermia. We observed aggregates of the thermally responsive ELP by fluorescence videomicroscopy within the heated tumor microvasculature but not in control experiments, which demonstrates that the phase transition of the thermally responsive ELP carrier can be engineered to occur in vivo at a specified temperature. By exploiting the phase transition-induced aggregation of these polypeptides, this method provides a new way to thermally target polymer-drug conjugates to solid tumors.

Amino Acid Sequence↗
PubMed2001

Enhanced TOF-SIMS imaging of a micropatterned protein by stable isotope protein labeling.

Patterning of biomolecules on surfaces is an increasingly important technological goal. Because the fabrication of biomolecule arrays often involves stepwise, spatially resolved derivatization of surfaces, spectroscopic imaging of these arrays is important in their fabrication and optimization. Although imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a powerful method for spatially resolved surface analysis, TOF-SIMS images of micropatterned proteins on organic substrates can be difficult to acquire, because of the lack of high intensity, protein-specific molecular ions that are essential for imaging under static conditions. In contrast, low-mass ions are of suitable intensity for imaging, but can originate from different chemical species on the surface. A potential solution to this problem is to utilize stable isotope labeled proteins, an approach that has heretofore not been explored in TOF-SIMS imaging of micropatterned proteins and peptides. To investigate the feasibility of stable isotope enhanced TOF-SIMS imaging of proteins, we synthesized 15N-labeled streptavidin by labeling of the protein during expression from a recombinant gene. The spatial distribution of streptavidin bound to biotin micropatterns, fabricated on a polymer and on a self-assembled monolayer on gold, was imaged by TOF-SIMS. Imaging of high-intensity, low-m/z secondary ions (e.g., C15N-) unique to streptavidin enabled unambiguous spatial mapping of the micropatterned protein with a lateral resolution of a few micrometers. TOF-SIMS imaging of micropatterned 15N-labeled streptavidin also illustrated the exquisite sensitivity of TOF-SIMS to low fractional coverage of protein (5 A effective thickness) in the background regions of the protein micropattern.

Image Processing, Computer-Assisted↗
PubMed2000

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acrylamides↗
PubMed2000

Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

Antibodies↗
PubMed1999

Direct force measurements of the streptavidin-biotin interaction.

The interaction between streptavidin and its ligand, biotin, were studied by direct force measurements. The complimentary approaches of surface force apparatus (SFA) and atomic force microscopy (AFM) were used to elucidate both long-range and short-range adhesive interactions of the streptavidin biotin interaction. The high spatial resolution of the SFA provided a detailed profile of the intersurface forces of apposing surfaces functionalized with streptavidin and biotin. Measurements obtained by the SFA corresponded to long and intermediate-range forces that are important in determining ligand receptor association. AFM was used to measure the unbinding force of individual streptavidin biotin complexes. These measurements revealed the short-range interactions (i.e. hydrophobic and hydrogen bonding forces) that stabilize the intermolecular bond.

Biophysics↗
PubMed1999

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Binding Sites↗
PubMed1999

X-ray crystallographic studies of streptavidin mutants binding to biotin.

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.

Binding Sites↗
PubMed1999

Purification of recombinant proteins by fusion with thermally-responsive polypeptides.

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.

Amino Acid Sequence↗
PubMed1998

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex.

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.

Binding Sites↗
PubMed1996

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Bacterial Proteins↗
PubMed1995

Control of protein-ligand recognition using a stimuli-responsive polymer.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Acrylic Resins↗
PubMed1995

Investigating the relationship between surface chemistry and endothelial cell growth: partial least-squares regression of the static secondary ion mass spectra of oxygen-containing plasma-deposited films.

The relationship between endothelial cell growth and surface properties of plasma-deposited films (PDFs) was investigated using partial least-squares regression (PLS). PDFs of oxygen-containing precursors were prepared under various conditions, and bovine arterial endothelial cells (BAECs) were grown on these substrates. Secondary ion mass spectrometry (SIMS) in the static mode was used to characterize the surface chemistry of these substrates. The growth of BAECs on the PDFs was correlated to the positive and negative static SIMS spectra of the PDFs by PLS. A good correlation between the SIMS spectra of PDFs and endothelial cell growth was obtained. Qualitative information was also extracted from the multivariate model, giving some information as to the most important variables influencing BAEC growth.

Animals↗
PubMed1995

Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120.

We report the functional characterization of site-directed biotin binding-site mutants of recombinant core streptavidin. The mutagenesis studies were aimed at characterizing the contributions of Trp residues known to contact biotin that have been postulated to control the exceptional binding affinity observed in this system. The functional properties of single site-directed mutants replacing Trp residues with Phe or Ala at positions 79, 108, and 120 were investigated by quantitating the EC50 binding parameters of these mutants to biotin and 2-iminobiotin in an ELISA format. The biotin EC50 for all mutants was the same as wild-type streptavidin, demonstrating that their delta Ka values relative to wild type were < 10(6). The conservative W79F and W108F mutants displayed only a 2- to 3-fold increase in EC50 for 2-iminobiotin, corresponding to an estimated delta Ka < 10, while the W120F mutant displayed a much greater alteration in 2-iminobiotin EC50, corresponding to an estimated delta Ka of 10(2). These delta Ka values are likely to reflect similar changes for biotin. The 2-iminobiotin EC50 values for the Ala mutants fell outside the accessible concentration range of the ELISA assay, demonstrating that these mutations lowered the Ka by a factor of 10(4) to 10(6). Direct estimation of biotin Ka values for W79A, W120A, and W120F in an ultrafiltration binding assay yielded Ka values of 4.3 x 10(7) M-1, 8.6 x 10(6) M-1, and > 5 x 10(9) M-1, respectively, in excellent agreement with the ELISA estimates of delta Ka with 2-iminobiotin as a reporter ligand. The results of these preliminary functional studies suggest that these aromatic side chains contribute significantly to the streptavidin-biotin binding free energy.

Bacterial Proteins↗