Recovery Of Lysozyme Research Articles

Separation of lysozyme from salted duck egg white by affinity precipitation using pH-responsive polymer with an l-thyroxin ligand

Lysozyme could be efficiently purified using affinity precipitation by using a pH-responsive polymer PMMDN with l-thyroxin as affinity ligand. A pH-responsive polymer PMMDN was polymerized and subsequently coupled with l-thyroxin as the ligand. The pI of the affinity polymer was 4.65 and the recovery was 96.7% of its original amount after recycling three times. The Optimal adsorption condition was 0.02M phosphate buffer (pH 5.5) with 1.0mol/L NaCl, and the adsorption isotherm showed the maximum adsorption capacity as 22.76mg/g polymer, the dissociation constant as 0.085mg/ml, and the label-free detection data analyzed by ForteBio’s Octet also verified the results. The recovery of total lysozyme by elution with 0.2mol/L Gly-NaOH buffer (pH 10.0), and the maximum elution recoveries were 94.32% (protein) and 96.79% (activity). The surface morphologies of the samples in the whole process of affinity precipitation were obtained by scanning electron microscope (SEM).

Comparison of Six Sample Preparation Methods for Analysis of Food Additives in Milk Powder

Milk powder matrix is highly complex with significant amounts of lipids and proteins; thus, the determination of trace residues and contaminants in it often requires extensive sample extraction and preparation prior to instrumental analysis. An ideal preparation method is simple, less time consuming, and relatively inexpensive. It enhances the recovery and involves low consumption of toxic organic solvents. In this study, six sample preparation methods for milk powder were established and compared, including liquid–liquid extraction, organic precipitation, heavy precipitation, and three different solid-phase extraction (SPE) methods. The extraction recovery was evaluated by determination of six preservatives and sweeteners (benzoic acid, sorbic acid, natamycin, lysozyme, saccharin sodium, and aspartame) by HPLC-DAD in whole and skimmed milk powder samples. The results showed that the reproducibility of liquid–liquid extraction was not as high as the SPE method although the recovery was beyond 80 %; the organic precipitation and heavy precipitation gave poor recovery (below 20 %) of lysozyme; the highest recovery (>85 %) was achieved using the HLB cartridge compared to C18 and MAX cartridges. Therefore, the HLB cartridge was selected as the most appropriate sample preparation material for the determination of the six preservatives and sweeteners in milk powder samples by HPLC-DAD.

PURIFICATION OF LYSOZYME BY INTRINSICALLY SHIELDED HYDROGEL BEADS

Macro-sized intrinsically shielded hydrogel beads have been prepared from BSA and CM-dextran grafted with CB using a technique based on freeze-thawing gelation method. The size of the beads lies in around 500 μm. Isothemal titration calorimetry (ITC) showed that the relative binding affinities of the lysozyme for CB, compared with BSA, at pH 3.0 was stronger than that at pH 7.4. They were employed for the affinity separation of lysozyme using chromatography column. Their adsorption capacity for lysozyme at pH 3.0 is higher than that at pH 9. In a binary mixture of lysozyme and ovalbumin, the beads showed very high selectivity toward lysozyme. Lysozyme of very high purity (> 93%) was obtained from a mixture of lysozyme and ovalbumin, and 85% from egg white solution. The results indicate that the macro-sized bead can be used for the separation, purification, and recovery of lysozyme in a chromatograph column.

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100kDa membrane but 20-30% of the extracted BvLz was lost. Concentration of clarified extracts using a 3kDa membrane resulted in twofold purification and 65% recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50% BvLz purity and 69% recovery of BvLz.

The optimization of aqueous two‐phase extraction of lysozyme from crude hen egg white using response surface methodology

AbstractBACKGROUND: Aqueous two‐phase extraction is a versatile method for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG 4000/potassium citrate aqueous two‐phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white.RESULTS: Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol 4000 (PEG 4000)/potassium citrate ATPS are established using response surface methodology. Screening experiments using fractional factorial designs show that the pH of the system significantly affects the recovery and purification of lysozyme. An optimized ATPS was proved to be at pH 5.5 and 30 °C and contained 18% (w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) potassium chloride (KCl). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were 31100 U mg−1, 21.11 and 103%, respectively.CONCLUSION: The PEG 4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme. © 2012 Society of Chemical Industry

Preparation of weak cation exchange packings for chromatographic separation of proteins using ``click chemistry''

"Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.

Acceleration of Disulfide-Coupled Protein Folding by Positively Charged Glutathione Derivatives

Protein folding is a spontaneous process under appropriate condition and results in the formation of the native tertiary structure. However, disulfide-containing proteins require a redox system to form the correct disulfide pairings. Glutathione plays an important role in the formation of disulfide bonds in proteins in the endoplasmic reticulum. Because of this, it is also widely used as a redox reagent in the disulfide-coupled folding of proteins. However, the mechanism of the disulfide-coupled folding of proteins by glutathione is not completely understood. Therefore, to examine the glutathione mediated-folding of proteins containing disulfide bonds, a series of glutathione derivatives, Glu-Cys-Arg (ECR) and Arg-Cys-Gly (RCG) were prepared and their participation in protein folding was examined using lysozyme and prouroguanylin as model proteins.The folding recovery of lysozyme using the oxidized and reduced forms of RCG was higher than that of the typically used glutathione redox system, while the results for ECR were similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin indicated that the folding velocity using the RCG system was approximately 3 fold faster than that of the glutathione system. Furthermore, folding experiments using only the oxidized form of RCG revealed that prouroguanylin was converted into the native conformation more efficiently by RCG, compared to glutathione. We therefore conclude that the RCG system is superior to the glutathione system for protein folding because of its ability to accelerate the disulfide exchange reaction and its ability to mediate the formation of native disulfide bonds in proteins. In this paper, we report on the use of the positively charged glutathione molecule in disulfide-coupled protein folding, which results in an improved folding recovery and permits the formation of the correct tertiary structure of the target proteins.

Protein recovery from surfactant precipitation

The recovery of lysozyme from an aqueous solution containing precipitated lysozyme-AOT complexes formed by the direct addition of sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) to a lysozyme solution was studied using both solvents, and a counterionic surfactant. Ethanol,methanol and solvent mixtures dissolved the surfactant precipitate and recovered lysozyme as a solid. Recovery efficiency and protein stability varied with the type of solvent used. An entirely different method of recovery was also evaluated using a counterionic surfactant: tri-octylmethylammonium chloride (TOMAC) which bound to AOT releasing lysozyme into solution.Complete recovery (100%) of lysozyme was achieved at a molar ratio of 2:1(TOMAC:AOT), and the original protein activity was maintained in the final aqueous phase.The recovered lysozyme retained its secondary structure as observed in circular dichroism(CD) spectra. Specific activity studies show that counterionic surfactant extraction does not alter the biological activity of the enzyme.

Design of Protein Crystallization Processes Guided by Phase Diagrams

Many proteins including pharmaceuticals and industrial enzymes, after separation and purification, are high-value-added products. In this study, a general framework based on solid liquid equilibrium (SLE) phase diagrams for the conceptual design of protein crystallization processes is proposed. First, the coordinates for phase diagrams involving proteins are defined. Second, relevant regions of the phase diagram are determined by experiments. Finally, process synthesis is performed by visualizing the process paths on the phase diagram. This approach is demonstrated with two case studies. One is the design of a lysozyme recovery process in which salt-lean lysozyme solids are recovered from a lysozyme–sodium chloride solution. The other is the design of a process for separating lysozyme and ovalbumin in the presence of ammonium sulfate. The former design is in agreement with patent literature, and the latter design was verified with experiments. Thus, the feasibility of designing protein crystallization pro...

Effect of the SA content of a novel thermo‐sensitive P(NIPAM‐co‐SA) copolymer on denatured lysozyme refolding in vitro

AbstractA novel hydrogel of P(NIPAM‐co‐SA) copolymer was synthesized by inverse suspension polymerization by adding sodium acrylate (SA) to improve the phase transition properties of poly(N‐isopropylacrylamide) (PNIPAM). The morphologies, size distribution and thermosensitive characteristics of gel particles were studied and the maximal swelling ratio and LCST (Lower Critical Solution Temperature) of gel particles increased obviously with the addition of SA comonomer. When the protein concentration was 250 μg/mL, the optimized refolding conditions of denatured lysozyme with P(NIPAM‐co‐SA) hydrogel were that operating at the temperature of 35°C and a urea concentration of 2M, in which the mass ratio of P(NIPAM‐co‐SA) hydrogel with 4% SA copolymerized to lysozyme was 10 : 1. Under the optimized conditions, the activity recovery of lysozyme increased to 76.5% assisted by P(NIPAM‐co‐SA) gel particles compared with 55.6% by simple dilution. When refolding finished, the gel particles could be removed and recovered easily and the activity recovery of lysozyme was still as high as 61.5% after reused for 5 batches. With the addition of different amounts of SA comonomer, the hydrophobicity of the copolymer could be varied. Then the copolymerized hydrogel inhibits protein molecules aggregation more effectively through the moderate hydrophobic interactions between copolymers and protein molecules in the course of lysozyme refolding compared with the presence of PNIPAM polymer. All results above demonstrate that the P(NIPAM‐co‐SA) is a cost effective additive with tunable hydrophobicity for application in the refolding of recombinant proteins expressed as inclusion bodies in vitro. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Acyl cystamine: Small‐molecular foldase mimics accelerating oxidative refolding of disulfide‐containing proteins

Based on the structural characteristic of Protein disulfide isomerases and DsbA that have hydrophobic regions around the active sites, hydrophobic alkyl tails are linked to cystamine to create new small molecular foldase mimics, acyl cystamine. Both the oxidizing power and oxidation specificity of cystamine are enhanced by n-octanoyl or n-hexanoyl tail. N-octanoyl and n-hexanoyl cystamine are very effective to facilitate oxidative protein refolding at strong reducing environments. In the presence of 0.42 mM DTT, the activity recovery of lysozyme is over 90% by 90-min refolding with 0.1 mM n-octanoyl cystamine and 0.1 mM cystamine as oxidant, while almost no activity is recovered with 0.2 mM GSSG by 160-min refolding. For the refolding of 0.2 mg/mL lysozyme, with 0.6 mM n-hexanoyl cystamine and 1.12 mM residual DTT as redox agents, the activity recovery reaches as high as 93% after refolding for only 20 min. For ribonuclease A (RNase A) refolding, with 0.4 mM n-hexanoyl cystamine and 1.30 mM DTT, the recovery of activity reaches as high as 90% within 3 h. Thus, with n-octanoyl or n-hexanoyl cystamine as the oxidants, the necessity to remove excess DTT in the reduced and denatured protein solutions can be greatly alleviated. With a moderate hydrophobicity, n-hexanoyl cystamine is promising for application in oxidative protein refolding at an extensive concentration range. It is observed that in the oxidative refolding of 0.2 mg/mL lysozyme and RNase A, only about half of n-hexanoyl cystamine is needed when compared to cystamine to achieve the same kinetic effect.

In this issue

AbstractExpression system for high‐throughput screening.Aerts et al., Eng. Life Sci. 2011, 11, 10–19.The bottleneck of directed evolution and optimization of biocatalytic enzymes is usually the screening of mutants in multiwell plates. Before a library can be screened, soluble expression of the enzymes has to be achieved. To reduce the amount of consumables and number of pipetting steps in high‐throughput screening, Wim Soetaert et al. developed a constitutive expression system comprising four constitutive promoters expressing the target protein at differing strengths. In this issue, they demonstrate the use of this expression system for expression of sucrose phosphorylase (SP) from a range of microorganisms in E. coli. They could show effective expression of all target proteins with different promoters and at varying temperatures. This new expression system will present an indispensable tool to optimize enzyme expression for small scale expression and high‐throughput screening……………10http://dx.doi.org/10.1002/elsc.201000065magnified imageMagnetic lysozyme separationEichholz et al., Eng. Life Sci. 2011, 11, 75–83.Magnetic separation is commonly used at lab‐scale. In order to apply the same concept at a larger scale or even for industrial separation processes, effective and economical procedures have to be provided. First, suitable process equipment has to be developed. Second, the magnetic particles have to be manufactured with a stable surface functionalization and long‐term stability for their reuse. In this issue, researchers from the Karlsruhe Institute of Technology (KIT), Germany, introduce a new magnetic filter that overcomes the capacity limitations of the current high‐gradient magnetic separation technology. Magnetic beads with a strong acid cation‐exchange surface functionalization are synthesized and the separation procedure is implemented in a single‐unit operation. They successfully demonstrate the working principle by selective recovery of lysozyme from hen egg white with a purification factor of PF=36 and a purity P=0.83.……………75http://dx.doi.org/10.1002/elsc.201000121magnified imageFermentation process supervisionGnoth et al., Eng. Life Sci. 2011, 11, 94–106.Highly reproducible fermentation processes are essential for the production of recombinant therapeutic proteins and demanded by regular authorities such as the FDA or EMEA. A major prerequisite for correct process control is that the measurement devices work correctly. Feedback control is the best way to keep the cultures on track. However, feedback controllers require accurate online values of the controlled variables. To assess whether the measurement signals are correct, process supervision techniques are required. In the case where a process failure has occurred and the incorrectly measured variables have been identified, automated fail‐safe techniques must be started. In this issue, colleagues from Halle, Germany, and Kaunas, Lithuania, compare different approaches of process supervision and fail‐safe routines in the same production process of a recombinant protein in E. coli. They assess different control mechanism and models and compare their advantages and disadvantages.……………94http://dx.doi.org/10.1002/elsc.201000114magnified image

Recovery of lysozyme from hen egg white by selective magnetic cake filtration

AbstractA new concept for the improvement of the downstream processing and purification is the so‐called magnetic separation. By using surface functionalized magnetic substrate particles, which selectively adsorb the target product, it can be directly separated out of a crude bioprocess stream. These methods are already used for analytical purposes, where only small amounts of functionalized particles are necessary. To apply the same concept at a larger scale, effective and economical procedures have to be provided. First, suitable process equipment has to be developed. Second, the magnetic particles have to be manufactured with a stable surface functionalization and long‐term stability for their reuse. Up to now mainly high‐gradient magnetic separation filter devices are applied for selective magnetic separation. They consist of a magnetic matrix in which the magnetic particles are trapped. In this work, a new magnetic filter is introduced that overcomes the capacity limitations of the current high‐gradient magnetic separation technology. The principle is demonstrated by selective recovery of lysozyme from hen egg white. Prior to the separation experiments magnetic beads with a strong acid cation‐exchange surface functionalization are synthesized. The separation procedure is implemented in only one unit operation. With the implementation of the displacement elution sequence lysozyme can be separated out of a hen egg white solution with a purification factor of PF=36 and a purity P=0.83.

Sustained and Extended Release with Structural and Activity Recovery of Lysozyme from Complexes with Sodium (Sulfamate Carboxylate) Isoprene/Ethylene Oxide Block Copolymer

The complexation of lysozyme and sodium (sulfamate carboxylate) isoprene/ethylene oxide (SCIEO) at pH = 7.4 and the release of lysozyme from the complexes in the presence of NaCl were investigated. Through electrostatic and hydrophobic interactions, lysozyme and SCIEO form stable complex nanoparticles. The complexation partially disturbs the structure of lysozyme. Some of the hydrophobic residues of lysozyme are exposed to bind with SCIEO. The complexation leads to loss of most of the lysozyme activity. In the presence of NaCl, lysozyme can be released from the complexes. The released lysozyme molecules recover their native structure and activity completely. In the condition of physiological pH and ionic strength, a sustained and extended release of lysozyme was achieved.

Improved Lysozyme Stability and Release Properties of Poly(lactide-co-glycolide) Implants Prepared by Hot-Melt Extrusion

To assess the feasibility of hot-melt extrusion (HME) for preparing implants based on protein/poly(lactide-co-glycolide) (PLGA) formulations with special emphasis on protein stability, burst release and release completeness. Model protein (lysozyme)-loaded PLGA implants were prepared with a screw extruder and a self-built syringe-die device as a rapid screening tool for HME formulation optimization. Lysozyme stability was determined using DSC, FTIR, HPLC and biological activity. The simultaneous effect of lysozyme and PEG loadings was investigated to obtain optimized formulations with high drug loading but low initial release. Lysozyme was recovered from implants with full biological activity after HME. The release from all implants reached the 100% value in 60-80 days with nearly complete enzymatic activity of the last fraction of released lysozyme. Pure PLGA implants with up to 20% lysozyme loading could be formulated without initial burst. The incorporation of PEG 400 reduced the initial burst at drug loadings in excess of 20%. A complete lysozyme recovery in active form with a burst-free and complete release from PLGA implants prepared by hot-melt extrusion was obtained. This is in contrast to many reported microparticulate lysozyme-PLGA systems and suggests the great potential of hot-melt extrusion for the preparation of protein-PLGA implants.

Lysozyme extraction from hen egg white in an aqueous two-phase system composed of ethylene oxide–propylene oxide thermoseparating copolymer and potassium phosphate

The recovery and purification of lysozyme from hen egg white has been investigated in an aqueous two-phase systems composed of thermoseparating random copolymers of ethylene oxide (EO), propylene oxide (PO) and potassium phosphate. In the primary extraction step lysozyme was satisfactorily partitioned to the top polymer-rich phase in a system composed of 40% (w/w) EO 50PO 50, 10% (w/w) potassium phosphate, and 0.85 M sodium chloride at pH 9.0, diluted 3-fold with crude egg white, where contaminating proteins were discarded in the bottom phosphate-rich phase. After the primary phase separation the upper EO 50PO 50 phase was removed and subjected to temperature-induced (65 °C) phase separation, which resulted in the partitioning of pure lysozyme to the top water phase. The separation system was found to be efficient in achieving the purification of lysozyme in a high yield of 85% and specific activity of 32,300 U/mg of protein, with a purification factor of 16.9 and a concentration of lysozyme in the water phase of 2.3 g/l in two extraction steps.

High-speed protein purification by adsorptive cation-exchange hollow-fiber cartridges

Novel cation-exchange adsorptive membranes were assessed according to their protein adsorption capacity and permeation flowrate. Maximum static adsorption capacities for the three main egg-white proteins, lysozyme, ovoalbumin and conalbumin, were 140, 88 and 66 mg/ml, respectively. However, membranes showed an inverse relationship between permeation flowrate and static protein adsorption capacity. Two size cartridges (membrane volume of 0.42 and 3.5 ml) were built using the selected membrane. An adsorptive cross-flow cartridge was tested to recover and purify lysozyme from an egg-white solution. Breakthrough curves developed using a pure lysozyme solution showed a dynamic-to-static capacity ratio of 0.6, which was reduced to 0.4 during lysozyme recovery from egg-white solution in cross-flow mode. Total process cycle for the enzyme recovery and purification was in the range of 10–15 min for both cartridges. In both cases high-purity lysozyme (95%) was recovered with a productivity of 150 g/(l h) and no size-exclusion effect was detected.

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone (AMC) and ion exchange chromatography (IEC) were integrated, thus a new refolding method, artificial molecular chaperone–ion exchange chromatography (AMC–IEC) was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC–IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC–IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC–IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

Role of a novel multifunctional excipient poly(ethylene glycol)- block-oligo(vinyl sulfadimethoxine) in controlled release of lysozyme from PLGA microspheres

This study investigated the effect of ion-pairing of anionic polyelectrolytes: our novel poly(ethylene glycol)- block-oligo(vinyl sulfadimethoxine) (PEG-OVSDM) and poly(ethylene glycol)- block-poly( l-aspartic acid) (PEG-PAA) with cationic lysozyme on retention of protein stability during emulsification. Soluble lysozyme recovery after exposure to the deleterious interface was 42–88% (when ion-paired with PEG-OVSDM, PEG-OVSDM concentration dependent) compared to only 30% for free lysozyme. PEG-OVSDM provided a higher stabilization of lysozyme than PEG-PAA (36–60%). Lysozyme when recovered in the aqueous phase and analyzed by chromatography, enzymatic assay, fluorescence, and mass spectrometry showed no significant physicochemical change when compared with a lysozyme standard. Lysozyme was incorporated into poly(lactide-co-glycolide) (PLGA) microspheres via the typical double emulsion method. Incorporation of lysozyme complexes led to a higher encapsulation efficiency and loading amount, and a lower incidence of insoluble lysozyme aggregates compared to the control microspheres containing lysozyme only. More significantly, ion-pairing was able to dramatically reduce the initial lysozyme release to 18% compared with 50% from control microspheres and provided an overall better control of protein release. PEG-PAA was less effective than PEG-OVSDM in controlling the release probably due to weaker interactions between this polyelectrolyte and lysozyme. Manipulation of such polyelectrolyte–protein complexation may play a role in protein-controlled delivery.