Optical switching of protein interactions on photosensitive–electroactive polymers measured by atomic force microscopy

The ability to switch the physico-chemical properties of conducting polymers opens up new possibilities for a range of new applications. Appropriately functionalised materials can provide routes to multi-modal switching, for example in response light and/or electrochemical stimuli; this capability is important in the field of bionics, wherein remote control of the properties of materials opens new possibilities. For example, the ability to actuate a film via photonic stimuli is particularly interesting as it facilitates the modulation of interactions between surface host binding sites and potential guest molecules. In this work, we studied two different poly-terthiophenes: one was functionalized with a spiropyran photoswitch (pTTh-SP) and the second with a non photoswitchable methyl acetate moeity (pTTh-MA). These substrates were exposed to several cycles of illumination with light of different wavelengths and the resulting effect studied with UV-vis spectroscopy, contact angle and atomic force microscopy (AFM). The AFM tips were chemically activated with fibronectin (FN) and the adhesion force of the protein to the polymeric surface was measured. The pTTh-MA (no SP incorporated) showed a slightly higher average maximum adhesion (0.96 ± 0.14 nN) than the modified pTTh-SP surface (0.77 ± 0.08 nN), but after exposure of the pTTh-SP polymer to UV, the average maximum adhesion of the pTTh-MC was significantly smaller (0.49 ± 0.06 nN) than both the pTTh MA and pTTh-SP. These results suggest that hydrophobic forces are predominant indetermining the protein adhesion to the films studied and that this effect can be photonically tuned. By extension, this further implies that it should be possible to obtain a degree of spatial and temporal control of the surface binding behaviour of certain proteins with these functionalised surfaces through photoactivation/ deactivation, which, in principle, should facilitate patterned growth behaviour (e.g. using masks or directional illumination) or photocontrol of protein uptake and release

Poly(amidoamine)-BSA conjugates synthesised by Michael addition reaction retained enzymatic activity

Polymer-protein conjugates are key to overcome some of the therapeutic protein limitations, including inefficient intracellular delivery. Poly(amidoamine)s are bioresponsive polyelectrolytes, which can form complexes with proteins and promote their delivery into the cytosol of cells. To investigate if conjugation would affect the activity of the protein, two poly(amidoamine)-BSA conjugates were synthesised using a “grafted to” method and Michael addition reaction. Following purification, the conjugates were characterised by electrophoresis, size exclusion chromatography (Mn(C1) = 140.7 kDa ; Mn(C2) = 218.6 kDa) and light scattering (Dh(C1) = 37.5 nm ; Dh(C2) = 75.1 nm). As a result of the conjugation with the cationic polymer, the conjugates had a positive zeta potential (?(C1) = +15.4 mV; ?(C2) = +20.2 mV). TNBS assays demonstrated that 16% to 25% of the protein amine groups were modified and HPLC analysis indicated that the amount of protein in the conjugate was 0.76 mg of BSA/mg of PAA (C1) and 0.43 mg of BSA /mg of PAA (C2). Enzymatic assays indicated the conjugates displayed an esterase activity similar (C1) or reduced ~ 35% (C2) compare to BSA. Altogether the results demonstrated that the conjugation of poly(amidoamine)s to a model protein can lead to the formation of bioconjugates that retain the enzymatic activity of the native protein. Such conjugates could have some application in protein delivery and enzyme engineering for biocatalysis and biosensors

Photo-switching of protein activities by conjugation of photo-responsive polymers to proteins

Thesis (Ph. D.)--University of Washington, 2001Photo-switching of protein functions has recently become more actively investigated by many researchers. Our objective is to create novel photo-switchable proteins by conjugating photo-responsive polymers to proteins site-specifically.We synthesized N,N-dimethylacrylamide (DMA)-co-phenylazophenylacrylate copolymer (DMAA) and DMA-co-phenylazophenylacrylamide copolymer (DMAAm) with vinylsulfone termini for conjugation to a protein containing a cysteine. These polymers exhibited temperature-induced phase transitions, and opposite and reversible photo-responses; DMAA became soluble, while DMAAm became insoluble upon the same UV light irradiation.The mutant streptavidins (SAs), E116C and S139C, were conjugated to these polymers site-specifically. The conjugates bound the ligand, biotin, below the phase transition temperature of the polymer, and released it above the phase transition temperature. The E116C-DMAA conjugate released biotin upon VIS light, while the E116C-DMAAm conjugate released it upon UV light. These opposite photo-responses corresponded to the opposite photo-induced phase transitions of the conjugated polymers. The E116C conjugate, which contains the conjugation site at a critical position for biotin-binding, exhibited larger responses than the S139C conjugate which contains it on the opposite side to biotin-binding. The conjugation of larger MW polymer and the addition of free polymer also increased the responses.The mutant endoglucanaseIII, S25C and N55C, were also conjugated to the polymers. The conjugates exhibited similar temperature- and photo-responsive activity changes to the SA conjugates. Polymers with higher MW and the addition of the free polymer enhanced the switching activity. The N55C conjugates, which locate the conjugated polymer closer to the catalytic site than S25C, exhibited higher switching activity than the S25C conjugates. Both conjugates exhibited less activity toward a large substrate, hydroxyethylcellulose, than toward a small substrate, o-nitrophenylcellobioside. Kinetic studies revealed that the activity shut-off stemmed from the sterical inhibition by the shrunken polymers.Therefore, the photo-switching of molecular recognition processes and catalytic activity were achieved by photo-responsive polymer-protein site-specific conjugation. The MW of the polymer, the position of conjugation, the size of the substrate and the addition of free polymer were key factors in the design of such molecular switches. This methodology can be utilized to develop novel photo-switchable proteins in the fields of biomedicine, bioprocesses, and bioelectronics

Synthesis and degradation test of hyaluronic acid hydrogels

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). H-1 NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS3), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests. (c) 2006 Elsevier B.V. All rights reserved.X1156sciescopu

Selectively crosslinked hyaluronic acid hydrogels for sustained release formulation of erythropoietin

A novel sustained release formulation of erythropoietin (EPO) was developed using hyaluronic acid (HA) hydrogels. For the preparation of HA hydrogels, adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and analyzed with H-1 NMR. The degree of HA-ADH modification was about 69%. EPO was in situ encapsulated into HA-ADH hydrogels through a selective cross-linking reaction of bis(sulfosuccinimidyl) suberate (BS3) to hydrazide group (pK(a) = 3.0) of HA-ADH rather than to amine group (pK(a) > 9) of EPO. The denaturation of EPO during HA-ADH hydrogel synthesis was drastically reduced with decreasing pH from 7.4 to 4.8. The specific reactivity of BS3 to hydrazide at pH = 4.8 might be due to its low pKa compared with that of amine. In vitro release of EPO in phosphate buffered saline at 37 degrees C showed that EPO was released rapidly for 2 days and then slowly up to 4 days from HA-ADH hydrogels. When the hydrogels were dried at 37 degrees C for a day, however, longer release of EPO up to 3 weeks could be demonstrated. According to in vivo release test of EPO from HA-ADH hydrogels in SD rats, elevated EPO concentration higher than 0.1 ng/mL could be maintained from 7 days up to 18 days depending on the preparation methods of HAADH hydrogels. There was no adverse effect during and after HA-ADH hydrogel implantation. (c) 2006 Wiley Periodicals, Inc.X11303sciescopu

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble–insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h–induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in ~75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins