Removal of proteases from <Emphasis Type="Italic">Clostridium perfringens</Emphasis> fermented broth by aqueous two-phase systems (PEG/citrate)

In order to reduce the toxicity of Clostridium perfringens fermentation broths used in vaccine preparation, we developed two-phase aqueous systems for removal of toxin-activating proteases. Removal of the proteases inhibits the conversion of protoxin to active toxin. In order to establish the conditions under which the phase separation occurs, binodal curves, formed by poly(ethylene glycol) (PEG) and sodium citrate, were investigated at different values of pH and PEG molar mass. A 2⁴-experimental design was used to evaluate the influence of PEG molar mass and concentration, citrate concentration and pH on protease partition coefficient, removal factor and protease removal yield. It has been found that simultaneous increase in PEG molar mass and decrease in citrate concentration remarkably improved the removal factor, whereas the protease removal yield showed an opposite trend. The best conditions for the system under consideration (removal factor of 2.69 and yield of 116%) were obtained at pH 8.0 using PEG molar mass of 8000 g mol⁻¹ and concentrations of PEG and citrate of 24 and 15%, respectively.     

Poly(ethylene glycol)-mediated molar mass control of short-chain- and medium-chain-length poly(hydroxyalkanoates) from Pseudomonas oleovorans

Three strains of Pseudomonas oleovorans, a well known poly(hydroxyalkanoate) (PHA) producer, were tested for the ability to control PHA molar mass and end group structure by addition of poly(ethylene glycol) (PEG) to the fermentation medium. Each strain of P. oleovorans - NRRL B-14682 (B-14682), NRRL B-14683 (B-14683), and NRRL B-778 (B-778) - synthesized a different type of PHA from oleic acid when cultured under identical growth conditions. Strain B-14682 produced poly(3-hydroxybutyrate) (PHB), while B-14683 synthesized a medium-chain-length PHA ( mcl-PHA) with a repeat unit composition ranging from C4 to C14 and some mono-unsaturation in the C14 alkyl side chains. Strain B-778 synthesized a mixture of PHB (95 mol%) and mcl-PHA (5 mol%). The addition of 0.5% (v/v) PEG (M(n) =200 g/mol, PEG-200) to the fermentation broth of strains B-14682 and B-778 resulted in chain termination through esterification at the carboxyl terminus of the PHB with PEG chain segments, thus reducing the molar mass by 54% and 23%, respectively. The molar mass of the mcl-PHA produced by strains B-14683 and B-778 also showed a 34% and 47% reduction in the presence of PEG-200, respectively, but no evidence of esterification was present. PEG-400 (M(n) =400 g/mol) had a reduced effect on PHA molar mass. In fact, the molar masses of the mcl-PHA derived from strain B-14683 and both the PHB and mcl-PHA from B-778 were unchanged by PEG-400. In contrast, the PHB produced by B-14682 showed a 35% reduction in molar mass in the presence of PEG-400.     

Size fractionation of double-stranded DNA by precipitation with polyethylene glycol

We show that DNA molecules of differing molecular mass are separable by selective precipitation with polyethylene glycol (PEG+.. Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA. Double-stranded DNA can be fractionated at least in the range of 3 times 10-7 to 1 times 10-5 daltons. The effects on PEG concentration, sodium chloride concentration, DNA concentration, pH, divalent ions, precipitation time, and centrifugal force have been determined. These studies show PEG precipitation offers a size fractionation method for DNA which is convenient, of high capacity, and applicable over a wide range of conditions. However, resolution is not high and separation of two species approaches 100% only if they differ in molecular mass by at least a factor of two.     

Effect of polyethylene glycol on the thermal stability of green fluorescent protein

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70–95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D‐values obtained were almost constant for temperatures of 85, 90, and 95°C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75°C, PEG 600 and 4,000 g/mol stabilized GFP. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Partitioning of glycomacropeptide in aqueous two-phase systems

The partition behavior of glycomacropeptide (GMP) was determined in polyethylene glycol (PEG) and sodium citrate aqueous two-phase systems (ATPS). It was found that the partitioning of GMP depends on PEG molar mass, tie line length, pH, NaCl concentration and temperature. The obtained data indicates that GMP is preferentially partitioned into the PEG phase without addition of NaCl at pH 8.0. Larger tie line lengths and higher temperatures favor GMP partition to the PEG phase. Furthermore, it was verified that PEG molar mass and concentration have a slight effect on GMP partition. The increase in the molar mass of PEG induces a reduction of the protein solubility in the top PEG rich phase, being shown that the use of PEG1500 is beneficial for the extraction of GMP. A protein recovery higher than 85% was obtained in the top phase of these systems, clearly demonstrating its suitability as a starting point for the separation of GMP.     

Noncovalent adducts of poly(ethylene glycols) with proteins

A new method of preparation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumine) under high pressure has been developed. The involvement of polymer in the complexes was proved using (3)H-labeled PEG. The composition of the complexes (the number of polymer chains per one ChT molecule) depends on the molecular mass of PEG and decreases with the increase in molecular mass from 300 to 4000, whereas the portion of the protein (wt %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt %. The kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical with the corresponding values for the native ChT. According to the data obtained by the method of circular dichroism, the enzyme in the complexes fully retains its secondary structure. The steric availability of PEG polymer chains in the complexes was evaluated by their complexation with alpha-cyclodextrin (CyD) or polymer derivatives of beta-CyD modified with PEG (PEG-beta-CyD). In contrast to free PEG, only part of PEG polymer chains ( approximately 10%) interact with alpha-CyD. Thus, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule located far from the active site and results in the sufficient decrease in the availability of polymer chains. The complexes between PEG chains in PEG-protein adducts and PEG-beta-CyD may be considered as a novel type of dendritic structures.     

Extractive fermentation of clavulanic acid by Streptomyces DAUFPE 3060 using aqueous two-phase system

The influence of four variables, specifically PEG molar mass (400, 1,000, and 8,000 g/mol), concentrations of PEG and phosphate salts (15, 20, and 25% for both), and agitation intensity (110, 150, and 200 rpm), on clavulanic acid (CA) extraction by extractive fermentation with PEG/phosphate salts aqueous two-phase system was investigated in shaken flasks using a 2(4-1) -fractional factorial design. After selection of the two most significant variables (agitation intensity and PEG molar mass), an optimization study conducted according to a 2(2) -central composite design revealed that 25% PEG 8,000 g/mol and phosphate salts at 240 rpm (run 6) were the best conditions for the extractive fermentation, leading to the best results in terms of partition coefficient (k = 8.2), yield of CA in the PEG-rich phase (η(T) = 93%) and productivity (P = 5.3 mg/Lh). As a first attempt to make a scale-up of these results, the effectiveness of the extractive fermentation was then checked in a bench-scale bioreactor under conditions as close as possible to the optimum ones determined in flasks. The highest CA concentration obtained in the PEG-rich phase (691 mg/L) was 30% higher than in flasks, thus demonstrating the potential of such a new process, integrating the production and extraction steps, as a promising, low-cost tool to obtain high yields of this and similar products.     

Polymorphic phase behavior of lysopalmitoylphosphatidylcholine in poly(ethylene glycol)-water mixtures

The polymorphic phase behavior of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine dispersions in excess water has been studied as a function of temperature and poly(ethylene glycol) (PEG) concentration, using proton dipolar-decoupled 31P NMR spectroscopy and turbidity measurements. The phase behavior was found to depend on both lipid concentration and PEG concentration, and most of the NMR experiments were conducted at a lipid concentration of 15 mg/mL. At low PEG concentrations (0-12 wt %), a thermotropic transition occurs at 3-5 degrees C with increasing temperature, from an interdigitated lamellar gel (L beta i) phase to a normal micellar phase. At intermediate PEG concentrations (12-20 wt %), thermotropic transitions take place with increasing temperature, first from the lamellar gel phase to a fluid cubic (Q alpha) phase and then at higher temperatures from the cubic phase to the micellar phase. At intermediate PEG concentrations above the former range (20-30 wt %), thermotropic transitions take place with increasing temperature, first from the lamellar gel phase to the cubic phase, then from the cubic phase to a normal hexagonal (HI) phase, and finally from the hexagonal phase to the micellar phase. At high PEG concentrations (greater than 30 wt %), a thermotropic transition takes place with increasing temperature from the lamellar gel phase directly to the fluid hexagonal phase. At these high PEG concentrations, the micellar phase is not attained within the accessible temperature range (less than or equal to 90 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of chain length and polymer concentration on the gelation of (amidated) low-methoxyl pectin induced by calcium

The gelation of low-methoxyl pectin (LMP) induced by addition of Ca2+ was studied by measuring the storage modulus as a function of temperature during cooling. Samples with different molar masses were prepared by mechanical degradation. The effect of the molar mass and the pectin concentration on the gelation properties was investigated. The effect of partial amidation was studied by comparing LMP and partially amidated LMP with the same molar mass and degree of methylation. The results are compared to those from a model developed for Ca2+-induced pectin gelation, and good agreement is found except at low concentrations and low molar masses where the gels are weaker than predicted. At low concentrations intrachain bonding weakens the gel, while the presence of small pectin chains weakens the gel because it neutralizes binding sites on larger chains.     

Partial Hydrolysis of Poly(2-ethyl-2-oxazoline) and Potential Implications for Biomedical Applications?

The hydrolysis of PEtOx is studied to evaluate the potential toxicity of partially hydrolyzed polymers that might interfere with its increasing popularity for biomedical applications. The hydrolysis of PEtOx is studied in the presence of digestive enzymes (gastric and intestinal) and at 5.8?M hydrochloric acid as a function of temperature (57, 73, 90, and 100?°C). It is found that PEtOx undergoes negligible hydrolysis at 37?°C and that thermal and solution properties are not altered when up to 10% of the polymer backbone is hydrolyzed. Mucosal irritation and cytotoxicity is also absent up to 10% hydrolysis levels. In conclusion, PEtOx will not decompose at physiological conditions, and partial hydrolysis will not limit its biomedical applications.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

Mesoscopic simulation of self-assembly of linear and dendritic copolymer poly(styrene)-b-poly(ethyleneglycol) in polar and non-polar solvents

In this work, we simulate the microphase separation of aqueous and non-aqueous solutions of diblock copolymer poly(styrene)-b-poly(ethyleneglycol) under different architectures (linear and linear–dendritic) by dissipative particle dynamics. The observed morphologies in water where poly(ethyleneglycol) (PEG) block is soluble are as follows: (1) at low concentrations spherical micelles, cylinders and bicontinuous structures are formed in dendritic structures and spheres, cylinders and perforated lamellas in linear structure. The architectures simulated at low–moderate concentrations show an evolution sphere → cylinder → bicontinuous or perforated lamellas as the concentration is increased. (2) At high concentrated solutions rich defect structures of the sponge type are formed. In a non-aqueous non-polar solution such as cyclohexane, which is a good solvent for the polystyrene block, the formation of well-defined aggregates at low concentrations is not observed; however, irregular structures are achieved in concentrated solutions. We compare these results with a polymeric chimera consisting of a mixture of linear poly(styrene) homopolymer and PEG homopolymer in the linear, G1 or G2 dendritic configurations. Our simulations are in agreement with the experimentally observed structures of these polymers.     

Effect of poly(ethylene glycol) on the Ca2+-induced fusion of didodecyl phosphate vesicles

This paper reports a study of the effect of the dehydrating agent poly(ethylene glycol) (PEG) on didodecyl phosphate (DDP) bilayers and on the fusion activity of DDP vesicles as a function of the molecular weight of PEG. PEG 8K in a concentration of 10 wt % does not induce fusion. However, Ca2+-induced fusion is promoted as reflected by a lowering of the Ca2+ threshold concentration. This effect can most likely be attributed to the dehydrating capacity of the polymer. Interestingly, low concentrations (0.1 wt %) of PEG 20 K induce a moderate fusion capacity. At higher concentrations (0.5 wt %) fusion is inhibited, irrespective of the presence of Ca2+. These molecular weight dependent effects can be rationalized by taking into account that the clouding temperature differs for PEGs of different molecular weights. In the case of PEG 20K a microscopic phase separation will occur at the bilayer-water interface because PEG-PEG interactions and presumably PEG-DDP interactions are favored over PEG-water interactions. As a consequence, the DDP vesicle surface becomes covered with PEG 20K, resulting in a steric stabilization of the vesicles. This will impede or prevent, depending on the polymer concentration, the vesicles from approaching each other sufficiently close for fusion to occur.     

Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies.

Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.     

Bilayer curvature and certain amphipaths promote poly(ethylene glycol)-induced fusion of dipalmitoylphosphatidylcholine unilamellar vesicles.

Unilamellar vesicles of varying and reasonably uniform size were prepared from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) by the extrusion procedure and sonication. Quasi-elastic light scattering was used to show that different vesicle preparations had mean (Z-averaged) diameters of 1340, 900, 770, 630, and 358 A (sonicated). Bilayer-phase behavior as detected by differential scanning calorimetry was consistent with the existence of essentially uniform vesicle populations of different sizes. The response of these different vesicles to treatment with poly(ethylene glycol) (PEG) was monitored using fluorescence assays for lipid transfer, contents leakage, and contents mixing, as well as quasi-elastic light scattering. No fusion, as judged by vesicle contents mixing and change in vesicle size, was detected for vesicles of diameter greater than 770 A. The diameters of smaller vesicles increased dramatically when treated with high concentrations of PEG, although mixing of their contents could not be detected both because of their small trapped volumes and because of the extensive leakage induced in small vesicles by high concentrations of PEG. Lipid transfer was detected between vesicles of all sizes. We conclude the high bilayer curvature does encourage fusion of closely juxtaposed membrane bilayers but that highly curved vesicles appear also to rupture and form larger structures when diluted from high PEG concentration, a process that can be confused with fusion. Despite the failure of PEG to induce fusion of large, uncurved vesicles composed of a single phosphatidylcholine, these vesicles can be induced to fuse when they contain small amounts of certain amphiphathic compounds thought to play a role in cellular fusion processes. Thus, vesicles which contained 0.5 mol % L-alpha-lysopalmitoylphosphatidylcholine, 5 mol % platelet activating factor, or 0.5 mol % palmitic acid fused in the presence of 30%, 25%, and 20% (w/w) PEG, respectively. However, vesicles containing 1,2-dipalmitoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, 1-oleoyl-2-acetyl-sn-glycerol, or monooleoyl-rac-glycerol at surface concentrations up to 5 mol % did not fuse in the presence or absence of PEG. There was no correlation between the abilities of these amphipaths to induce phase separation or nonlamellar phases and their abilities to support fusion of pure DPPC unilamellar vesicles in the presence of high concentrations of PEG. The results are discussed in terms of the type of disrupted lipid packing that could be expected to favor PEG-mediated fusion.     

Electrophoretic mobility of human red blood cells coated with poly(ethylene glycol)

Poly(ethylene glycol), abbreviated as PEG, was covalently attached to the surface of human red blood cells (RBC) and the effects of such coating on the regions near the cell's glycocalyx were explored by means of cell electrophoresis. RBC electrophoretic mobilities were measured, in polymer-free buffers of various ionic strengths, as functions of PEG molecular mass (3.35, 18.5, 35.0, 35.9 kDa), geometry, (linear or 8-arm branched) and polymer/RBC ratio during attachment. The results indicate marked decreases of the mobility (up to 85%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment only removes positively-charged amino groups on the cell membrane, such decreases of mobility likely reflect structural changes near and within the RBC glycocalyx. Experimental results were analyzed using an extended "hairy sphere" model to consider friction and thickness of the polymer layer. Calculated polymer layer thickness increased with molecular mass for linear PEGs and was less extended for a branched PEG of similar molecular mass. Friction within the polymer layer increased with polymer/RBC ratio and for the linear PEGs was inversely related to molecular mass; friction was greatest for the branched PEG. Our results are consistent with the effects of attached PEGs on RBC aggregation and surface antigenic site masking, and suggest the usefulness of electrophoretic mobility techniques for studies of bound neutral polymers.     

Size‐selective DNA separation: Recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required

In the presence of sodium chloride (NaCl), DNA fragments can be size‐selectively separated by varying the final concentration of polyethylene glycol (PEG). This separation strategy in combination with the use of paramagnetic particles provides a valuable platform for achieving the desired DNA size interval, which is important in automated library preparation for high‐throughput DNA sequencing. Here, we report the establishment of recovery spectra of DNA fragments that enable the determination of suitable NaCl and PEG concentrations for size‐selective separation. Firstly, at a given NaCl concentration, the recovery equation was obtained by fitting the DNA recovery ratios versus the PEG concentrations using the logistic function to determine the required parameters. Secondly, the slope function of the recovery equation was achieved by deducing its first derivative. Therefore, the recovery spectrum can be generated using the slope function based on those parameters. According to the recovery spectra of different length DNA fragments, suitable NaCl and PEG concentrations can be determined, respectively, by calculating their resolution values and recovery ratios. The strategy was effectively applied to the size‐selective separation of 532‐, 400‐, and 307‐bp fragments at the selected reagent concentrations with recoveries of 96.9, 64.7, and 85.9%, respectively. Our method enables good predictions of NaCl and PEG concentrations for size‐selective DNA separation.     

Depletion interactions and modulation of DNA‐intercalators binding: Opposite behavior of the “neutral” polymer poly(ethylene‐glycol)

In this work we have investigated the role of high molecular weight poly(ethylene‐glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single‐molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an “inert polymer” with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non‐neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA‐ligand interactions. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 227–233, 2016.     

Poly(ethylene glycol)-induced lipid mixing but not fusion between synthetic phosphatidylcholine large unilamellar vesicles

We have examined the effect of poly(ethylene glycol) (PEG) on stable large unilamellar vesicles formed by a rapid extrusion technique and composed of pure synthetic phosphatidylcholines. The lipid systems studied were the saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the monounsaturated 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). PEG at all concentrations (3.8-40 wt %) induced lipid mixing between large vesicles composed of these phosphatidylcholines. Extensive leakage of internal contents also occurred at high PEG concentrations. However, in contrast to our previous report [Parente, R. A., & Lentz, B. R. (1986) Biochemistry 25, 6678], we could detect no mixing of internal contents indicative of fusion. This discrepancy is due to environmental factors that affect the behavior of 8-amino-naphthalene-1,3,6-trisulfonic acid (ANTS), the fluorophore used in the assay for contents mixing and leakage [McIntyre, Parks, Massenburg, & Lentz (1991) (submitted)]. In agreement with the results of the fusion assay, quasielastic light-scattering measurements revealed no increase in vesicle size following treatment with PEG. These results emphasize the importance of using assays for both membrane mixing and contents mixing to demonstrate fusion, since significant lipid mixing occurred in the absence of fusion. We conclude that large vesicles composed of pure phosphatidylcholine do not fuse in the presence of even high concentrations of PEG. However, DOPC vesicles containing a small amount of an amphipathic "impurity" have been shown to fuse in the presence of PEG at 23 degrees C. These results are discussed in terms of their implications for the mechanism of PEG-induced membrane fusion.     

Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2)

Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.