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

A 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     

Bulk Crystallization of Proteins by Low‐Pressure Water Evaporation

An advanced technique for bulk protein crystallization based on solvent evaporation is introduced. Hen egg white lysozyme is crystallized from its buffered aqueous solution in a stirred tank at low pressure and, thus, moderate boiling temperatures. No crystallizing agent to reduce the solubility is needed for crystallization. The biological activity of the enzyme remains unchanged during the process and a high crystal yield is achieved. Tetragonal and rod‐like lysozyme crystals, presumably of orthorhombic type, are crystallized at different boiling temperatures. This crystallization technique is proposed as a promising option to crystallize proteins economically, controllably, and gently. This method may replace standard salt‐out steps in existing production processes.     

Rapid Matrix‐Assisted Refolding of Histidine‐Tagged Proteins

Matrix refolded : The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in‐solution, this parallelized, matrix‐assisted approach allows the refolding of various proteins in a fast and efficient manner.

     

Hen Egg‐White Lysozyme Crystallisation: Protein Stacking and Structure Stability Enhanced by a Tellurium(VI)‐Centred Polyoxotungstate

As synchrotron radiation becomes more intense, detectors become faster and structure‐solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson–Evans‐type polyoxometalate (POM), specifically Na₆[TeW₆O₂₄] ? 22 H₂O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg‐white lysozyme (HEWL), which co‐crystallises with TEW in the vicinity (or within) the liquid–liquid phase separation (LLPS) region. The X‐ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry‐related protein chains. Thus, TEW facilitates the formation of protein–protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation.     

Crystallization of Hen Egg White Lysozyme by Solvent Freeze‐Out: Effect of Cooling Rate on Protein Inclusion in the Ice Layer

A new technique for crystallizing proteins is introduced. Solvent removal by freezing‐out is employed to crystallize hen egg white lysozyme from aqueous salt solutions. The crystallization is carried out at moderate salt concentrations (1–10 wt % NaCl) and at pH 4.4–5.2. The required supersaturation for nucleation and crystal growth is achieved by removal of the solvent resulting in a concentration increase with respect to salt, buffer, and protein. A simple tube and shell heat exchanger is used to generate the driving force for crystallization of the ice. The enzymatic activity of the resulting crystals is measured after their dissolution and shows large variations with respect to the crystallization conditions, though no systematic behavior is observed. The effect of the cooling rate applied to the cold finger upon the protein concentration in the ice is investigated.     

Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)- lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(- 1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(- 1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N- terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg- white lysozyme.      

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

BACKGROUND: 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     

Partitioning of chicken egg white proteins in polyelectrolyte/salt aqueous two‐phase systems composed of polyethyleneoxide–maleic acid copolymer and potassium phosphate

Aqueous two‐phase systems were formed from solutions of a polyelectrolyte, polyethyleneoxide–maleic acid copolymer, and potassium phosphate. The properties of such aqueous two‐phase systems were highly dependent on pH. This was reflected in the partition behavior of three chicken egg white proteins: lysozyme, conalbumin and ovalbumin. Separability of these three proteins was improved by the use of the polyelectrolyte, in comparison with when uncharged polyethylene glycol (poly(1,2‐dihydroxyethane)) was used as a phase‐forming polymer. © 2002 Society of Chemical Industry     

Preparation of Zn2+‐chelated poly(HEMA‐MAH) cryogel for affinity purification of chicken egg lysozyme

Supermacroporous poly(2‐hydroxyethyl methacrylate) [poly(HEMA)]‐based monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with N‐methacryloyl‐L ‐histidine methyl ester (MAH) as functional comonomer and N,N′‐methylene‐bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for affinity purification of lysozyme from chicken egg white. The monolithic cryogel containing a continuous polymeric matrix having interconnected pores of 10–50 μm size was loaded with Zn²⁺ ions to form the metal chelate with poly(HEMA‐MAH) cryogel. Poly(HEMA‐MAH) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA‐MAH) monolithic cryogel was 5.62 g H₂O/g cryogel. Poly(HEMA‐MAH) cryogel containing 45.8 μmol MAH/g was used in the adsorption/desorption of lysozyme from aqueous solutions. The nonspecific adsorption of lysozyme was very low (7.5 mg/g). The maximum amount of lysozyme adsorption from aqueous solution in phosphate buffer was 209 mg/g at pH 7.0. It was observed that lysozyme could be repeatedly adsorbed and desorbed with the poly(HEMA‐MAH) cyogel without significant loss of adsorption capacity. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Simultaneous refolding,purification, and immobilization of recombinant Fibrobacter succinogenes 1,3‐1,4‐β‐D‐glucanase on artificial oil bodies

BACKGROUND: 1,3‐1,4‐β‐D‐glucanase (1,3‐1,4‐β‐D‐glucan 4‐glucanohydrolase; EC 3.2.1.73) has been used in a range of industrial processes. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of 1,3‐1,4‐β‐D‐glucanase was investigated. RESULTS: A 1,3‐1,4‐β‐D‐glucanase gene from Fibrobacter succinogenes was overexpressed in Escherichia coli as a recombinant protein fused to the N terminus of oleosin, a unique structural protein of seed oil bodies. With the reconstitution of the artificial oil bodies (AOBs), refolding, purification, and immobilization of active 1,3‐1,4‐β‐D‐glucanase was accomplished simultaneously. Response surface modeling (RSM), with central composite design (CCD), and regression analysis were successfully applied to determine the optimal temperature and pH conditions of the AOB‐immobilized 1,3‐1,4‐β‐D‐glucanase. The optimal conditions for the highest immobilized 1,3‐1,4‐β‐D‐glucanase activity (7.1 IU mg^?1 of total protein) were observed at 39 °C and pH 8.8. Furthermore, AOB‐immobilized 1,3‐1,4‐β‐D‐glucanase retained more than 70% of its initial activity after 120 min at 39 °C, and it was easily and simply recovered from the surface of the solution by brief centrifugation; it could be reused eight times while retaining more than 80% of its activity. CONCLUSIONS: These results indicate that the AOB‐based system is a comparatively simple and effective method for simultaneous refolding, purification, and immobilization of 1,3‐1,4‐β‐D‐glucanase. Copyright © 2009 Society of Chemical Industry     

一种温敏性水凝胶复性体系:聚N-异丙基丙烯酰胺的制备及其在溶菌酶复性中的应用

Temperature-sensitive hydrogel-poly(N-isopropyl acrylamide) (PNIPA) was prepared and applied to protein refolding. PNIPA gel disks and gel particles were synthesized by the solution polymerization and inverse suspension polymerization respectively. The swelling kinetics of the gels was also studied. With these prepared PNIPA gels, the model protein lysozyme was renatured. Within 24 h, PNIPA gel disks improved the yield of lysozyme activity by 49.3% from 3375.2 U.mg-1 to 5038.8 U.mg-1. With the addition of faster response PNIPA gel beads,the total lysozyme activity recovery was about 68.98% in 3h, as compared with 42.03% by simple batch dilution.The novel refolding system with PNIPA enables efficient refolding especially at high protein concentrations. Discussion about the mechanism revealed that when PNIPA gels were added into the refolding buffer, the hydrophobic interactions between denatured proteins and polymer gels could prevent the aggregation of refolding intermediates,thus enhanced the protein renaturation.     

CTAB辅助溶菌酶复性过程动力学

研究了溶菌酶在十六烷基三甲基溴化铵（CTAB）溶液中的复性过程,通过测定溶液表面张力与酶活力的变化证实变性溶菌酶首先与CTAB形成复合物,进而在氧化-还原剂作用下开始复性并与CTAB发生解离.非还原型SDS-PAGE分析结果表明,复性反应的主要产物有三类：具有天然结构的溶菌酶单体、溶菌酶多聚体及溶菌酶单体与CTAB形成的无活性复合物.不同产物的含量取决于溶液中CTAB与溶菌酶的摩尔比.当变性溶菌酶浓度为0.1～0.4mg•ml^-1,CTAB与溶菌酶摩尔比为5～20时,采用“变性-复性”二态复性模型分析了CTAB辅助溶菌酶复性过程的宏观动力学.结果表明,随CTAB与溶菌酶摩尔比的增大,变性反应的速率常数显著增大,而折叠反应的速率常数先增大后减小,CTAB与溶菌酶的摩尔比为10时,复性率最高.而蛋白质浓度提高则导致折叠反应速率常数减小,变性反应速率常数增大,复性率缓慢下降.     

An Oxidative Bioconjugation Strategy Targeted to a Genetically Encoded 5‐Hydroxytryptophan

Approaches that enable the chemoselective, covalent modification of proteins in a site‐specific manner have emerged as a powerful technology for a wide range of applications. The electron‐rich unnatural amino acid 5‐hydroxytryptophan was recently genetically encoded in both Escherichia coli and eukaryotes, thereby allowing its site‐specific incorporation into virtually any recombinant protein. Herein, we report the chemoselective conjugation of various aromatic amines to full‐length proteins under mild, oxidative conditions that target this site‐specifically incorporated 5‐hydroxytryptophan residue.     

The molecular weight ratio of monomethoxypolyethylene glycol (mPEG) to protein determines the immunotolerogenicity of mPEG proteins

Immunotolerogenic activity of monomethoxypolyethylene glycol-(mPEG) conjugated proteins is a beneficial property in proteinpharmaceutics. However, procedures for the preparation of tolerogenicmPEG proteins have not yet been defined. We prepared mPEG proteinswith different mPEG contents using three proteins, hen egg lysozyme,ovalbumin and bovine gamma globulin, and their tolerogenicitiesto antigen-specific T and B cell responses were examined. Wefound the most appropriate ratio of tolerance induction to be1.5–2.0, which is the molecular weight ratio of conjugatedtotal mPEGs to protein. This value may assist in the preparationof tolerogenic mPEG proteins.     

座滴法制备鸡蛋白溶菌酶晶体与生长因素探究

蛋白质晶体学的不断发展,使得蛋白质晶体的结晶方法也日趋增多。首次采用了座滴法结晶法,探究不同浓度鸡蛋白溶菌酶溶液,不同温度、NaCl浓度,是否添加mPEG对晶体形貌尺寸的影响。通过显微镜以及扫描电镜观察蛋白质晶体形貌特征,不同条件下的晶体形貌变化,最终获得晶体生长的最适温度以及浓度条件。     

Lysophosphatidic Acid Produced by Hen Egg White Lysophospholipase D Induces Vascular Development on Extraembryonic Membranes

Lysophosphatidic acid (lysoPtdOH), a lysophospholipid mediator, exerts diverse physiological effects, including angiogenesis, through its specific G‐protein‐coupled receptors. Previously, we showed that unfertilized hen egg white contains polyunsaturated fatty acid‐rich lysoPtdOH and lysophospholipase D (lysoPLD). Here, we examined whether lysoPtdOH was produced by lysoPLD in the presence and absence of a hen fertilized ovum and what the physiological role of lysoPtdOH in hen egg white is. Mass spectrometry showed that fertilized hen egg white contained about 8 μM lysoPtdOH before incubation with an ovum, mainly comprised of 18:1‐ (12.6 %), 18:2‐ (37.8 %) and 20:4‐molecular species (41.5 %). In an early gestation period, the lysoPtdOH was increased up to 9.6 μM, concomitant with a decrease in the level of polyunsaturated lysophosphatidylcholine (lysoPtdCho). Moreover, lysoPtdOH‐degrading activities were found in egg white and the vitelline membrane, showing that these enzymes control lysoPtdOH levels in egg white. In an egg yolk angiogenesis assay, two lysoPtdOH receptor antagonists, Ki16425 and N‐palmitoyl serine phosphoric acid (NASP), inhibited blood vessel formation induced by exogenously added 18:1‐lysoPtdOH and its precursor lysoPtdCho on the hen yolk sac. Ki16425 and NASP also inhibited blood vessel formation in the chorioallantoic membrane (CAM). Furthermore, the relatively higher levels of LPA₁, LPA₂, LPA₄ and LPA₆ mRNA were present in the yolk sac and CAM. These results suggest that lysoPtdOH produced from lysoPtdCho by the action of lysoPLD in hen egg white is involved in the formation of blood vessel networks through several lysoPtdOH receptors on various extraembryonic membranes, including the yolk sac membrane and CAM.     

Influence of Urea and Dimethyl Sulfoxide on K-Peptide Fibrillation

Protein fibrillation leads to formation of amyloids—linear aggregates that are hallmarks of many serious diseases, including Alzheimer’s and Parkinson’s diseases. In this work, we investigate the fibrillation of a short peptide (K-peptide) from the amyloidogenic core of hen egg white lysozyme in the presence of dimethyl sulfoxide or urea. During the studies, a variety of spectroscopic methods were used: fluorescence spectroscopy and the Thioflavin T assay, circular dichroism, Fourier-transform infrared spectroscopy, optical density measurements, dynamic light scattering and intrinsic fluorescence. Additionally, the presence of amyloids was confirmed by atomic force microscopy. The obtained results show that the K-peptide is highly prone to form fibrillar aggregates. The measurements also confirm the weak impact of dimethyl sulfoxide on peptide fibrillation and distinct influence of urea. We believe that the K-peptide has higher amyloidogenic propensity than the whole protein, i.e., hen egg white lysozyme, most likely due to the lack of the first step of amyloidogenesis—partial unfolding of the native structure. Urea influences the second step of K-peptide amyloidogenesis, i.e., folding into amyloids.     

Secretion of heterologous and native proteins,growth and morphology in batch cultures of Aspergillus niger B1‐D at varying agitation rates

The influence of bioreactor operational conditions on the micromorphology of batch cultures of Aspergillus niger B1‐D, containing a hen egg white lysozome (HEWL) marker protein, was examined using computerised image analysis. Significant differences in micromorphology were observed with increased stirrer speed, with shorter organisms with shorter hyphal elements occurring as agitation speed increased, even though mean tip numbers were similar. This may explain the observed increase in the total extracellular protein, since the ratio of synthetic (tip) to non‐synthetic zones became increasingly favourable. HEWL concentrations fell above 500 rpm, probably due to the effects of DOT (dissolved oxygen tension) on the glucoamylase HEWL fusion. HEWL was susceptible to proteolytic degradation, by native proteases, during the autolytic phase. Such insights may indicate why a gene from one mould expressed in a close relative can give production levels equivalent to levels of native enzymes, while secretion of a gene from a ‘distant’ source, eg a higher eukaryotic gene, occurs at much lower levels. © 1999 Society of Chemical Industry     

Modeling and control of protein crystal shape and size in batch crystallization

In this work, the modeling and control of a batch crystallization process used to produce tetragonal hen egg white lysozyme crystals are studied. Two processes are considered, crystal nucleation and growth. Crystal nucleation rates are obtained from previous experiments. The growth of each crystal progresses via kinetic Monte Carlo simulations comprising of adsorption, desorption, and migration on the (110) and (101) faces. The expressions of the rate equations are similar to Durbin and Feher. To control the nucleation and growth of the protein crystals and produce a crystal population with desired shape and size, a model predictive control (MPC) strategy is implemented. Specifically, the steady‐state growth rates for the (110) and (101) faces are computed and their ratio is expressed in terms of the temperature and protein concentration via a nonlinear algebraic equation. The MPC method is shown to successfully regulate both the crystal size and shape distributions to different set‐point values. © 2013 American Institute of Chemical Engineers AIChE J, 59: 2317–2327, 2013     

Hydrogels based on copolymers of 2‐hydroxyethylmethacrylate and 2‐hydroxyethylacrylate as a delivery system for proteins: Interactions with lysozyme

Hydrogels have attracted considerable attention due to numerous applications, in particular as contact lenses and carriers for sustained drug delivery. The aim of the present work is to characterize the interactions of copolymer hydrogels consisted of 2‐hydroxyethylmethacrylate (HEMA) and 2‐hydroxyethylacrylate (HEA) with a small protein (lysozyme) and to assess the potential applications of these hydrogels as a drug delivery system for sustained release of protein‐based therapeutics. Physicochemical properties of protein‐loaded hydrogels, as well as lysozyme in vitro loading and release and the conformation of the protein released from hydrogels were studied. The effect of copolymer composition on the protein deposition on hydrogels and protein aggregation in the presence of hydrogels was also assessed. The results show that introduction of HEA into the copolymeric hydrogels enhances their suitability as a delivery system for proteins. Copolymerisation of HEMA and HEA allows controlling the physicochemical properties of hydrogels and the protein release rate. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44768.