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The strategy of using polymer-grafted media is effective to create protein chromatography of high capacity and uptake rate, giving rise to an excellent performance in high-throughput protein separation due to its high dynamic binding capacity. Taking the scientific development and technological innovation of protein chromatography as the objective, this review is devoted to an overview of polymer-grafted media reported in the last five years, including their fabrication routes, protein adsorption and chromatography, mechanisms behind the adsorption behaviors, limitations of polymer-grafted media and chromatographic operation strategies. Particular emphasis is placed on the elaboration and discussion on the behaviors of ion-exchange chromatography (IEC) with polymer-grafted media because IEC is the most suitable chromatographic mode for this kind of media. Recent advances in both the theoretical and experimental investigations on polymer-grafted media are discussed by focusing on their implications to the rational design of novel chromatographic media and mobile phase conditions for the development of high-performance protein chromatography. It is concluded that polymer-grafted media are suitable for development of IEC and mixed-mode chromatography with charged and low hydrophobic ligands, but not for hydrophobic interaction chromatography with high hydrophobic ligands and affinity chromatography with ligands that have single binding site on the protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Polymer-grafted media have been a focus of recent development for ion exchange chromatography (IEC) because of their capacity and uptake kinetics that can lead to high dynamic capacity in protein purification. This work is devoted to developing novel cation exchangers of high adsorption performance by grafting polymerization of sodium methacrylate (MA) onto a commercial agarose gel Sepharose FF (FF). Five polyMA (pMA)-grafted FF gels were prepared with the same grafting density but different chain lengths (i.e., different ionic capacities, ICs), and named as FF-pMA-IC (IC denotes IC value in mmol/L). The effects of chain length (IC) and ionic strength (IS) on protein adsorption and chromatographic behaviors were examined using lysozyme (at pH 8.0) and γ-globulin (at pH 5.0) as model proteins. It was found that lysozyme adsorption capacity increased with increasing IC till reaching a plateau (390 mg/mL) over IC=540 mmol/L (FF-pMA-540), while there was an optimum IC (320 mmol/L, FF-pMA-320) at which γ-globulin adsorption capacity reached the highest (208 mg/mL). With increasing chain length (IC), the uptake rates of both the proteins presented decreasing trends due to the steric hindrance caused by the polymer chains. At the same IC, however, the uptake rate of lysozyme was much higher than that of γ-globulin because of the different sizes of the two proteins. Increasing salt concentration obviously promoted the uptake rates of the proteins, which led to the increase of dynamic binding capacities (DBCs) in different salt concentration ranges. The DBC value of lysozyme on FF-pMA-540 kept as high as 108-198 mg/mL in the salt concentration range of 0-150 mmol/L, and the DBC of γ-globulin on FF-pMA-320 increased to 27 mg/mL with increasing salt concentration from 100 mmol/L. This work clearly indicated the presence of optimal IC values (chain lengths) for different sized proteins, and IS was also crucial for reaching a high DBC for a specific protein. The findings provided insight into the selection of FF-pMA-n gels and operational conditions (e.g., IS) for the purification of a target protein of defined size., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Polymer-grafting to porous materials is an effective way to create protein ion-exchangers of high capacity and uptake rate because the 3D architecture of the polymeric ion exchange groups provides high binding space and facilitated transport of the bound protein. Herein, a new anion exchanger was fabricated by grafting (3-acrylamide propyl) trimethyl ammonium chloride (APTAC) onto Sepharose FF gel for protein adsorption and chromatography. The ion exchanger, denoted as FF-pAPTAC, presented high capacity but limited uptake rate in bovine serum albumin (BSA) adsorption due to the high charge density of the cationic polymer chains. To solve the problem, we proposed to copolymerize APTAC with an electroneutral monomer, acrylamide (AM), onto Sepharose FF to modulate the charge density of the grafted polymer chains. By decreasing the feeding molar ratio of APTAC to AM, the ionic capacity (IC) of the copolymerized resins, FF-p(AM-APTAC)n (n denotes IC in mmol/L), decreased, but the chain length could be remained almost unchanged due to the similar reactivity ratios of the two monomers. With decreasing IC, the static adsorption capacity (q m ) of FF-p(AM-APTAC)n decreased gradually because of the decline of protein binding sites. The uptake rate, however, represented by the ratio of effective pore diffusivity to the free solution diffusivity (D e /D 0 ), exhibited a strong uptrend with decreasing IC, reaching ~2.5-fold of the maximum observed with FF-pAPTACn. It is considered that the decrease of charge density weakened protein binding strength to the chains and increased the chain flexibility, which consequently facilitated the transport of bound proteins on the chains. Moreover, at the same IC, each FF-p(AM-APTAC)n displayed similar adsorption capacity but high uptake rate as compared with its FF-pAPTACn counterpart mainly due to the longer chain length of the copolymer. Particularly, ~6.5-fold enhancement of D e /D 0 was observed at IC = 50 ± 2 mmol/L. Both high adsorption capacity and uptake rate made FF-p(AM-APTAC)n exhibit superior dynamic binding performance. The findings proved that reducing chain charge density by copolymerizing an electroneutral monomer was promising for fabrication of high-performance protein ion exchangers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Our previous studies on protein adsorption to the anion-exchangers of poly(ethylenimine) (PEI)-grafted Sepharose FF found that both adsorption capacity and uptake rate of bovine serum albumin (BSA) increased greatly when the PEI grafting density reached over a critical ionic capacity (cIC) due to the 3D protein binding and occurrence of "chain delivery" of bound proteins. Moreover, by the investigation on the anion-exchangers of diethylaminoethyl (DEAE)-modified and DEAE-dextran-grafted Sepharose FF, we found the unique role of surface ligand in facilitating protein uptake kinetics on dextran-functionalized anion exchangers as the "transfer station" for the "chain delivery" of bound proteins. However, what would be the contribution of surface ligands on the transport of bound proteins on PEI chains at a wide range across cIC, particularly at the lower IC range where no "chain delivery" was present? We have thus designed this research to answer the question. To this purpose, we fabricated three series of PEI-Sepharose FF resins, one without surface ligand (i.e., FF-PEI) and two with surface DEAE modifications at DEAE coupling densities of 60 and 90 mmol/L (i.e., FF-D60-PEI and FF-D90-PEI), and focused on the role of surface DEAE at different PEI densities in BSA adsorption equilibrium and uptake kinetics in a wide IC range of PEI across cIC (∼600 mmol/L for BSA). It was found that, at low grafting-ligand densities (IC cIC, however, both adsorption capacity and uptake rate changed only slightly (<5% and <10%, respectively) by the addition of surface DEAE groups. The results revealed that at IC < cIC, the surface DEAE groups provided the supplement of available binding sites and facilitated the happenings of the "chain delivery" of bound proteins as the "transfer station", both of which were limited at the low IC range. However, at IC > cIC, the extended flexible PEI chains have already afforded 3D binding space with high accessibility and easy happening of "chain delivery" of bound proteins, so the surface DEAE groups between neighboring PEI chains did not work in assisting the transport of bound proteins. Moreover, when the IC was lower but close to the cIC, the FF-D-PEI resins exhibited higher uptake rates than FF-PEI resins at a similar or even lower IC values. These findings indicate that surface modification of charged groups in PEI-based anion exchangers of low grafting density would be an efficient strategy to fabricate high performance protein ion exchangers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diabetic nephropathy (DN) is one of the common diabetic complications, but the mechanisms are still largely unknown. In this study, we constructed a DN related protein-protein interaction network (DNPPIN) on the basis of RNA-seq analysis of renal cortices of DN and normal mice, and the STRING database. We analyzed DNPPIN in detail revealing nine critical proteins which are central in DNPPIN, and contained in one network module which is functionally enriched in ribosome, nucleic acid binding and metabolic process. Overall, this study identified nine critical and functionally associated protein-coding genes concerning DN. These genes could be a starting point of future research towards the goal of elucidating the mechanisms of DN pathogenesis and progression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Lens fibrosis involves aberrant growth, migration, and transforming growth factorβ (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). In this study, we investigated the role of the bromo- and extra-terminal domain (BET) inhibitor in lens fibrotic disorder to identify drug-based therapies., Methods: Rat lens explants, rabbit primary lens epithelial cells (rLECs), human lens explants and human SRA01/04 cells were treated with TGFβ2 in the presence or absence of the BET bromodomain inhibitor JQ1 or the MYC inhibitor 10058-F4. Proliferation was determined by MTS assay. Cell migration was measured by wound healing and transwell assays. The expression levels of fibronectin (FN), α-smooth muscle actin (α-SMA), E-cadherin, and phosphorylated downstream Smads were analyzed by Western blot, qRT-PCR, and immunocytochemical experiments. Transcriptome analysis was conducted to explore the molecular mechanism., Results: Blockage of BET bromodomains with JQ1 significantly suppressed rLECs proliferation by inducing G1 cell cycle arrest. Furthermore, JQ1 attenuated TGFβ2-dependent upregulation of mesenchymal gene expression and phosphorylation of Smad2/3 during the progression of EMT, whereas E-cadherin expression was preserved. JQ1 repressed MYC expression, which was dose- and time-dependently upregulated by TGFβ2. Inhibiting MYC with either the small-molecule inhibitor 10058-F4 or genetic knockdown phenocopied the effects of JQ1 treatment. MYC overexpression partially reversed the JQ1-regulated EMT-related alteration of gene expression. Both JQ1 and 10058-F4 blocked the expression of TGFβ receptor II and integrin αv in rLECs and abolished TGFβ2-induced opacification and subcapsular plaque formation in rat lens explants., Conclusions: Our results demonstrate the antifibrotic role of JQ1 in maintaining the epithelial characteristics of LECs and blocking TGFβ2-induced EMT, possibly by downregulating MYC, thereby providing new avenues for treating lens fibrosis.

The elaborate modulation of the transforming growth factor β (TGF-β) superfamily signaling network plays an essential role in tooth morphogenesis and differentiation. In our previous studies, we have demonstrated that TGF-β1 promotes enamel mineralization and maturation using TGF-β1 gene conditional knockout (TGF-β1-cKO) mice. However, the specific regulatory mechanisms of TGF-β1 during enamel development remain unclear. Furthermore, we have previously found that the expression of WD repeat-containing protein 72(WDR72)in mouse enamel epithelium is decreased significantly in the absence of TGF-β1. Therefore, the aim of the present study was to investigate how TGF-β1 affects amelogenesis by regulating the expression of Wdr72. Histological examination showed that the absence of TGF-β1 in ameloblastic epithelial cells resulted in a reduction in enamel mineralization and a delay in enamel matrix protein absorption. TGF-β1, Runt-related transcription factor 2(RUNX2) and WDR72 were revealed to be colocalized in ameloblasts by immunohistochemistry, and it was also found that the expression of Runx2 and Wdr72 was markedly different between TGF-β1-cKO mice and wild type(TGF-β1-WT)mice. In addition, the effect of exogenous TGF-β1 on Wdr72 was more significant when RUNX2 was present than when RUNX2 was absent. Furthermore, we found that there were binding sites for RUNX2 on the promoter of Wdr72 and that Wdr72 expression was regulated by RUNX2. Collectively, our results suggest that TGF-β1 affects enamel mineralization by modulating RUNX2 and thus affecting the expression of Wdr72., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

High recognition selectivity has been the main object in developing protein-imprinted materials. Here, we demonstrate a novel strategy for the controlled synthesis of PEGylated surface protein-imprinted nanoparticles with reduced nonspecific binding, which is based on sequential two steps of surface-initiated reversible addition-fragmentation chain transfer aqueous precipitation polymerization (SI-RAFT APP). Click chemistry was employed to construct hydrophilic nanocores with both high-density RAFT chain transfer agents and template-capturing groups. Through the first-step SI-RAFT APP, protein-imprinted nanoshells were formed over the nanocores using lysozyme as a model template. By the second-step SI-RAFT APP, nonlinear PEG chains were grafted from the core-shell imprinted nanoparticles before the removal of the template. Both the thickness of the imprinted nanoshells and the length of the grafted chains could be readily controlled by the polymerization time. Thus the obtained PEGylated core-shell particles exhibited greatly improved template binding selectivity compared with the non-PEGylated controls, typically with the imprinting factor increasing from 2.1 to 9.1. Meanwhile, the PEGylation process did not impair but significantly enhance the protein binding capacity. The generality of the established approach was preliminarily proved by imprinting another template protein, bovine hemoglobin. This work represents the first example for the controlled synthesis and post-imprinting functionalization of surface protein-imprinted nanoparticles via SI-RAFT polymerization.

Ion exchangers of high binding capacity and uptake rate are desired for high performance purification of proteins by ion exchange chromatography. For this purpose, we have herein developed a new anion exchanger by grafting N,N-Dimethylaminopropyl acrylamide (DMAPAA) onto Sepharose FF by atom transfer radical polymerization. Six DMAPAA-grafted Sepharose FF resins of the same grafting density but different chain lengths (ionic capacities, ICs) were prepared, and named as FF-pDMAPAAn (n denotes IC value, 65-831 mmol/L). Bovine serum albumin (BSA) adsorption and its chromatography were studied to evaluate the anion exchange resins. It was found that the equilibrium protein adsorption capacity (q m ) increased rapidly from 91 mg/mL at IC = 65 mmol/L till reaching a peak value (368 mg/mL) at IC = 458 mmol/L and then decreased slightly with further increasing IC (chain length) to 831 mmol/L. The uptake rate, D e /D 0 , defined as the ratio of effective diffusion coefficient (D e ) to free diffusivity of the protein (D 0 ), also presented similar changes as a function of IC. Namely, D e /D 0 increased from 0.25 to 2.45 in the IC range of 65 to 458 mmol/L, kept almost unchanged from 458 to 573 mmol/L and then decreased slightly with further increase of IC to 831 mmol/L. Therefore, FF-pDMAPAA458 presented the best performance of the six resins for BSA adsorption, possessing very high q m and D e /D 0 values (368 mg/mL and 2.45, respectively). The resin was thus extensively characterized in the effect of ionic strength and column chromatography. The q m and D e /D 0 of FF-pDMAPAA458 were much higher than other anion exchangers reported in literatures and kept over 230 mg/mL and 2.3, respectively, at salt concentrations up to 100 mmol/L. Therefore, the dynamic binding capacity of the FF-pDMAPAA458 column was remarkably higher than Q Sepharose FF and other two typical polymer-grafted anion exchangers reported in literatures in the salt concentration range, and kept as high as 180-220 mg/mL in the superficial flow velocity rang of 150-1350 cm/h. Taken together, the results demonstrated that FF-pDMAPAA resins of appropriate ionic capacities are promising for high-performance protein chromatography., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Previously, we have studied protein adsorption behaviors on a series of poly(ethylenimine) (PEI)-grafted Sepharose FF, and a critical ionic capacity of PEI-grafted resins was observed, above which both protein adsorption capacity (q) and effective pore diffusivity (D e ) increased drastically. Moreover, reducing the charge density of the PEI-grafted resins from an ionic capacity of 740 mmol/L (FF-PEI-L740) to 440 mmol/L (FF-PEI-R440) by neutralization of the amino groups of PEI chains with sodium acetate brought out a three-fold increase of D e value at pH 8. In this work, FF-PEI-L740 and FF-PEI-R440 were selected to investigate the complicated effects of pH on protein adsorption behavior using bovine serum albumin (BSA) as the model protein. It was found that, for FF-PEI-L740, both the q and D e values decreased significantly when pH decreased from 9 to 5.5, and then increased dramatically at pH 5, and finally decreased remarkably at pH 4.5. The results were considered due to the following causes: The decrease of pH from 9 to 5.5 led to a greatly increased electrostatic repulsion between the PEI chains due to the increased dissociation degree of PEI, which caused the increase in steric hindrance effects and decrease in the bound protein transport by chain delivery. At pH 5, which was near the pI of BSA (∼4.9), the presence of few protein charges greatly decreased the electrostatic hindrance effect on protein transport and the high charge density of FF-PEI-L740 provided extensively accessible binding sites and facilitated the happening of chain delivery as well. At pH 4.5, the net charge of BSA shifted to positive, likely charged with PEI, so the overall electrostatic repulsion greatly hindered protein uptake. For FF-PEI-R440, whose charge density was much lower than FF-PEI-L740, its q and D e values even increased mildly when pH decreased from 8 to 5.5, because its slightly-increased charge density did not cause much increased electrostatic and steric hindrance effects but provided more binding sites for protein adsorption. At pH 5, the few charges of BSA and the low charge density of FF-PEI-R440 could not afford enough electrostatic interaction for protein binding, resulting in significantly decreased q and D e values. Besides, column breakthrough experiments revealed that FF-PEI-R440 kept high dynamic binding capacity (DBC) (>120 mg/mL) at pH 5.5-8, while FF-PEI-L740 offered high DBC (>80 mg/mL) at pH 5, 7 and 8 with a higher salt concentration (100 mmol/L NaCl). These findings demonstrated the excellence of FF-PEI-L740 and FF-PEI-R440 in different conditions, and would help in the design and selection of suitable resins for high-performance protein chromatography., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This work reports poly(allylamine) (PAA), as a polymeric ion-exchange ligand for protein chromatography. Sepharose FF was modified with PAA, and six anion exchangers with ionic capacities (ICs) from 165 to 618mmol/L were prepared. Inverse size exclusion chromatography, adsorption equilibrium, uptake kinetics and column elution were performed. It was found that both the adsorption capacity and effective diffusivity maintained low values in the IC range of 165-373mmol/L, but they started to increase beyond 373mmol/L, and increased by 80% and 23 times, respectively, when the IC reached 618mmol/L. Interestingly, a drastic decrease of pore size was observed around the IC of 373mmol/L. The results suggest that the PAA chains played an important role in protein adsorption by altering the inner pore structure of the gels. It is considered that, PAA chains turn from inextensible states with multipoint-grafting on the pore surface at low coupling densities (IC<373mmol/L) to closer, extended and flexible grafting states with less coupling points at higher coupling densities (IC>373mmol/L). These characters of the grafted chains at higher IC values benefit in protein adsorption by three-dimensional binding and encouraged the happening of "chain delivery" of bound proteins on the chains. Besides, the ion exchangers showed favorable adsorption and uptake properties in a wide ionic strength range, 0-500mmol/L NaCl, indicating much better salt tolerance feature than the so-far reported ion exchangers. Moreover, a mild condition of pH 5.0 offered effective recovery of bound proteins in elution chromatography. The results indicate that the PAA-based anion exchanger of a high IC value is promising for high-capacity protein chromatography dealing with feedstock of a wide range of ionic strengths., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Molecular imprinting is a promising way for constructing artificial protein recognition materials, but it has been challenged by difficulties such as restricted biomacromolecule transfer in the cross-linked polymer networks, and reduced template-monomer interactions that are due to the required aqueous media. Herein, we propose a strategy for imprinting of histidine (His)-exposed proteins by combining previous approaches such as surface imprinting over nanostructures, utilization of metal coordination interactions, and adoption of aqueous precipitation polymerization capable of forming reversible physical crosslinks. With lysozyme as a model template bearing His residues, imprinted polymer nanoshells were grafted over vinyl-modified nanoparticles by aqueous precipitation copolymerization of a Cu(2+) chelating monomer with a temperature-responsive monomer carried out at 37 °C, above the volume phase-transition temperature (VPTT) of the final copolymer. The imprinted nanoshells showed significant temperature sensitivity and the template removal could be facilitated by swelling of the imprinted layers at 4 °C, below the VPTT. The resultant core-shell imprinted nanoparticles exhibited strikingly high rebinding selectivity against a variety of nontemplate proteins. An imprinting factor up to 22.7 was achieved, which is among the best values reported for protein imprinting, and a rather high specific binding capacity of 67.3 mg/g was obtained. Moreover, this approach was successfully extended to preliminary imprinting of hemoglobin, another protein with accessible His. Therefore, it may be a versatile method for fabrication of high-performance surface-imprinted nanoparticles toward His-exposed proteins.

In the previous studies on protein adsorption to poly(ethylenimine) (PEI)-grafted Sepharose FF resins, a critical ionic capacity (600mmol/L) of PEI-Sepharose resins was found for the adsorption of bovine serum albumin (BSA), above which both protein capacity and uptake rate increased drastically. In this work, the influence of counterions on the PEI-Sepharose resin with an ionic capacity of 683mmol/L (FF-PEI-L680) was investigated with sodium salts of SCN(-), Cl(-), HPO4(2-) and SO4(2-). Linear gradient elution, batch adsorption and breakthrough experiments showed that counterion preference, effective pore diffusion coefficient (De) and dynamic binding capacity (DBC) values increased in the order of SCN(-), Cl(-), HPO4(2-) and SO4(2-), while static adsorption capacity decreased in this order. It is considered that higher counterion preference of the ion exchange groups resulted in lower protein binding strength and adsorption capacity, while the De value increased due to the enhanced "chain delivery" effect (a kind of surface diffusion). Besides, the DBC value was mainly dependent on De value. In particular, SO4(2-) was the most favorable counterion for the PEI-Sepharose resin, which gave rise to the highest De value (De/D0=1.17, D0 is protein diffusivity in free solution) and DBC value (118mg/mL at a residence time of 2min). Moreover, the effects of counterions on BSA adsorption to DEAE Sepharose FF and Q Sepharose FF, which were non-grafted resins, were also studied for comparisons. It was found that the counterion preferences of the two non-grafted resins were different from each other and also different from that of FF-PEI-L680. The different counterion preferences were attributed to the differences in the ion-exchange ligand chemistries. In addition, the De values for DEAE Sepharose FF and Q Sepharose FF kept unchanged. The low counterion sensitivity of De values could be interpreted as the lack of "chain delivery" effect for the non-grafted resins. The results indicate that protein adsorption and chromatographic performance with PEI-Sepharose can be improved by proper counterions. For the four counterions tested, SO4(2-) was the most favorable for providing the best adsorption and elution outcomes with FF-PEI-L680., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chromatography is the key technology in protein purification as well as in protein refolding. Taking the scientific development and technological innovation of protein chromatography as the objective, this article is devoted to an overview of protein behavior at chromatographic surfaces, including protein orientation, conformational transitions (unfolding and refolding), and protein transport. Recent advances achieved by using molecular simulations as well as theoretical and experimental investigations are elaborated and discussed with emphasis on their implications to the rational design of novel chromatographic surfaces or materials and mobile phase conditions for the development of high-performance protein chromatography., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Silica nanoparticles (SNPs) were sequentially modified with poly(ethylenimine) (PEI) and 2-diethylaminoethyl chloride (DEAE) to prepare a series of positively charged SNPs-PEI and SNPs-PEI-DEAE. The sequential double-modification strategy produced a charge density as high as 1740 μmol/g (4524 μmol/mL), which offered a very high adsorption capacity for bovine serum albumin (314 mg/g). Most importantly, the highly charged SNPs-PEI and SNPs-PEI-DEAE could efficiently facilitate the refolding of like-charged protein at extremely low utilization. For instance, in the refolding of 1 mg/mL lysozyme, the refolding yield reached 75% with only 3.3 μL/mL SNPs-PEI-DEAE. The bead consumption was reduced by nearly 96% as compared to that of the charged microspheres used previously to reach a similar yield. The results proved that the polyelectrolyte-modified SNPs were promising for applications in facilitating like-charged protein refolding, and the research opened up a new way for biotechnology applications of highly charged nanoparticles.

This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aqueous two-phase micellar systems (ATPMSs) composed of nonionic surfactants are considered promising for the separation and purification of proteins. To improve the specificity of ATPMSs, a novel nickel-chelated surfactant was prepared by successive modifications of Triton X-114 (TX). Characterizations by Fourier transformation infrared spectroscopy demonstrated the successful synthesis of the nickel-chelated surfactant (TX-Ni). The cloud point, critical micelle concentration (CMC), molecular interaction parameter and micelle size were measured for the mixed surfactant system of TX-Ni and TX to achieve a full understanding of their aggregation behaviors. The results showed that mixed micelles were formed, and the cloud point increased with the mole fraction of TX-Ni because TX-Ni had a more hydrophilic head group than TX. Moreover, the reduction of micelle size revealed by light scattering experiments indicated that the insertion of TX-Ni inhibited the micellar growth due to the increased steric and electrostatic repulsion. Finally, the efficiency of TX-Ni as an affinity surfactant was demonstrated by the affinity partitioning of histidine-tagged enhanced green fluorescent protein with an over 20-fold increase of the partition coefficient (from 0.60 to 12.42). This affinity-based ATPMS is thus considered promising for providing a versatile platform for the separation of histidine-rich proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Composite cryogel monoliths based on poly(2-hydroxyethylmethacrylate) (pHEMA) were fabricated by incorporating polymeric resin particles. The monoliths were sequentially modified by polyethylenimine (PEI) and diethylaminoethyl (DEAE). The novel composite material had rough pore walls and extended anion-exchange tentacles, which provided more binding sites for protein molecules. The dynamic adsorption capacity of bovine serum albumin (BSA) on the novel cryogel bed reached 11.2mg/mL bed volume at a flow velocity of 8cm/min, which was about 1.5-4.6 times higher than the cryogel beds obtained by single modifications. The capacity value was also much higher than the BSA capacities of cryogel beds reported in literature (1-6mg/mL). The capacity decreased only slightly with increasing flow rate from 0.6 to 12cm/min. The height equivalent to a theoretical plate of the composite beds was in the range 2-2.5mm, changed indistinctively in a flow rate range 0.6-18cm/min. Hence, the work has proved that the double-modification strategy was promising for enhancing protein adsorption capacity of cryogel monolith for high-speed protein chromatography., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To explore the details of protein uptake to polymer-grafted ion exchangers, Sepharose FF was modified with poly(ethylenimine) (PEI) to prepare anion exchanger of 10 different ionic capacities (ICs, 100-1220mmol/L). Adsorption equilibria and kinetics of bovine serum albumin (BSA) were then studied. It is found that ionic capacity, i.e., the coupling density of PEI, had significant effect on both adsorption capacity (qm) and effective protein diffusivity (De). With increasing ionic capacity, the qm value increased rapidly at IC<260mmol/L and then increased slowly till reaching a plateau at IC=600mmol/L. In the IC range of 100-600mmol/L, however, the De values kept at a low level (De/D0<0.07); it first decreased from 0.05±0.01 at IC=100mmol/L to 0.01±0.01 at IC=260mmol/L and then increased to 0.06±0.01 at IC=600mmol/L. Thereafter, sharp increases of the qm and De values [36% (from 201 to 273mg/mL) and 670% (from 0.06±0.01 to 0.49±0.04), respectively] were observed in the narrow range of IC from 600 to 740mmol/L. Finally, at IC>740mmol/L, the qm value decreased significantly while the De value increased moderately with increasing the IC. The results indicate that PEI chains played an important role in protein adsorption and transport. In brief, there was a critical IC (cIC) or PEI chain density, above which protein adsorption and transport behaviors changed drastically. The cIC was identified to be about 600mmol/L. Estimation of PEI grafting-layer thickness suggests that PEI chains formed an extended three-dimensional grafting-layer at IC>cIC, which provided high flexibility as well as accessibility of the chains for protein binding. Therefore, at IC>cIC, the adjacent PEI chains became close and flexible enough, leading to facilitated transport of adsorbed protein molecules by the interactions of neighboring chains mediated by the bound molecules. It is regarded as "chain delivery" effect. At the same time, improved accessibility of binding sites led the significant increase of binding capacity. The decrease of qm value at IC>740mmol/L is considered due to the decrease of effective porosity. The research has thus provided new insight into protein adsorption and transport in polymer-grafted ion-exchange media., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We have previously found that addition of like-charge media in a refolding system can greatly enhance protein refolding yield, and the media of higher charge density (q) are favorable for facilitating protein refolding. Herein, a double-modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (PGMA) microspheres (0.8μm). The PGMA beads were firstly modified with poly(ethylenimine) of molecular weight 60,000 (PEIL) or 1200 (PEIS) and then further modified with 2-diethylaminoethylchloride (DEAE) or PEIS. The charge density and bovine serum albumin (BSA) adsorption density (QBSA) of the beads were significantly increased by the double modification. The QBSA of the double-modified beads with the highest q (824μmol/g) reached 36.3mg/g, over 35% higher than that of the single-modified beads (26.7mg/g) with the highest q (300μmol/g) that a single modification could achieve. The double-modified beads with different ligand structures and charge densities were used for facilitating the refolding of like-charged lysozyme. Lysozyme refolding yield with the single-modified beads of the highest q (300μmol/g) was 60% at a critical bead concentration (cc) of 100mg/mL. By contrast, the refolding yields with the double-modified beads of q>500μmol/g were 70%, and the refolding yields with the double-modified beads of q≥650μmol/g could even reach 70% at a cc of 40mg/mL. This indicates that the double-modified beads of high q values could enhance like-charged protein refolding more significantly than the single-modified beads at a low bead utilization. The facilitating effect of the like-charged beads was independent of the ligand structure. The beads of higher q values showed lower salt sensitivity in protein refolding, and were expected beneficial for use in refolding buffers of higher ionic strengths. The sequential modification strategy for enhancing charge density would help develop more efficient media for protein adsorption and protein refolding applications., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Agarose-based matrices have been widely used in ion exchange chromatography (IEC). We have herein observed that positively charged proteins (lysozyme and cytochrome c) are adsorbed on the agarose-based anion-exchangers (Q and DEAE Sepharose FF gels) in a capacity of 10-40 μg/mL. In contrast, negatively charged protein (bovine serum albumin) is not adsorbed to Sepharose FF and SP Sepharose FF gels. Elemental analysis of the gel indicated that the residual anionic sulfate groups in agarose would have worked as the cation exchange groups for the positively charged proteins. The trace adsorption behavior of lysozyme onto Sepharose FF and Sepharose FF-based anion exchangers was studied and the effects of NaCl concentration and cation group density on the adsorption were examined for better understanding of the trace adsorption in chromatographic processes. At NaCl concentrations less than 0.05 mol/L, which is the normal adsorption condition in IEC, the trace adsorption kept at a high level, so this trace adsorption cannot be avoided in the ionic strength range of routine IEC operations. Grafting poly(ethylenimine) (PEI) chain of 60 kDa to a cation group density of 700 mmol/L could reduce the adsorption capacity to about 20 μg/mL, but further reduction was not possible by increasing the cation group density to 1200 mmol/L. Therefore, attentions need to be paid to the phenomenon in protein purification practice using agarose-based matrices. The research is expected to call attentions to the trace adsorption on agarose-based matrices and to the importance in the selection of the suitable solid matrices in the production of high-purity protein products in large-scale bioprocesses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have previously found that addition of charged particles in a refolding solution can greatly increase the refolding yield of like-charged proteins. Herein, porous anion exchangers of different charged group densities, ligand chemistries, pore sizes and particle sizes were prepared with Sepharose FF gel for studying their effects on the oxidative refolding of like-charged lysozyme. We found that charge density had significant contribution to the enhancing effects on lysozyme refolding. At low resin concentration range (<0.04-0.1g/mL), the refolding yield increased with increasing charged group density and resin concentration. The yield then reached a plateau at a critical resin concentration; the higher the charge density, the lower the critical resin concentration. This implies that gel particles of higher charge density were favorable to offer higher refolding yield at lower added concentrations. In the gel concentration range in which refolding yield has reached plateau, there existed an optimum charge density that gave the highest refolding yield. It was attributed to the electrostatic repulsion effect of the charged groups on the like-charged protein, which reduced the accessible pore volume for the protein. At the same charge density, the refolding yield was independent of ligand chemistry, but a polyelectrolyte group of higher molecular weight was more suitable for grafting the gel to prepare matrices of high charge density. The resins of smaller size exhibited better facilitating effect, and the microporous resin was better than that with superpores. The research is expected to help design more effective charged materials for facilitating protein refolding., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high-efficiency protein refolding. We have herein found that electrostatic repulsion between like-charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion-exchange resins when the protein and ion-exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like-charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies., (Copyright © 2010 Wiley Periodicals, Inc.)

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., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Epistasis (i.e. gene-gene interaction) has long been recognized as an important mechanism underlying the complexity of the genetic architecture of human traits. Definitions of epistasis range from the purely molecular to the traditional statistical measures of interaction. The statistical detection of epistasis usually does not map onto or easily relate to the biological interactions between genetic variations through their combined influence on gene expression or through their interactions at the gene product (i.e. protein) or DNA level. Recently, greater high-dimensional data on protein-protein interaction (PPI) and gene expression profiles have been collected that enumerates sets of biological interactions. To better align statistical and molecular models of epistasis, we present an example of how to incorporate the PPI information into the statistical analysis of interactions between copy number variations (CNVs). Among the 23 640 pairs of known human PPIs and the 1141 common CNVs detected among HapMap samples, we identified 37 pairs of CNVs overlapping with both genes of a PPI pair. Two CNV pairs provided sufficient genotype variation to search for epistatic effects on gene expression. Using 47 294 probe-specific gene expression levels as the outcomes, five epistatic effects were identified with P-value less than 10(-6). We found a CNV-CNV interaction significantly associated with gene expression of TP53TG3 (P-value of 2 × 10(-20)). The proteins associated with the CNV pair also bind TP53 which regulates the transcription of TP53TG3. This study demonstrates that using PPI data can assist in targeting statistical hypothesis testing to biological plausible epistatic interaction that reflects molecular mechanisms.

Agarose gels were fabricated by water-in-oil emulsification with the addition of CaCO(3) granules at 8-16 wt%. Thus agarose beads of different superporosities were produced after dissolving the solid porogen. The superporous agarose (SA) and homogeneous agarose gels were double cross-linked and modified with diethylaminoethyl chloride to produce anion exchangers. We have proposed to use a superporous replica (porous titania microspheres) to examine the superporous structure and pore size distribution of the soft gel. The replica was prepared with the agarose gel entrapping CaCO(3) granules by a sol-gel-templating method. It was found that the superpores created by CaCO(3) granules were uniformly distributed and ranged from 0.95 microm to 1.33 microm. The physical properties of the gels were significantly affected by the porogen content. Importantly, by increasing the solid porogen to 12 wt%, the bed permeability and effective porosity increased about 48% and 33%, respectively. Further increase in the porogen to 16 wt% led to a decrease of the mechanical strength. With increasing superpores in the beads, the dynamic adsorption capacity of the packed columns increased obviously at 305-916 cm/h. Besides, the column efficiency changed less with increasing flow velocity up to 1200 cm/h. It was concluded that the use of 12 wt% CaCO(3) granules in agarose solution was beneficial for the fabrication of the SA gel with good mechanical stability and promising performance for protein chromatography., (Copyright 2010 Elsevier B.V. All rights reserved.)

Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. Ligand density has significant effects on protein adsorption behavior, but little is understood about the effect of ligand homogeneity on surface morphology of ligands, protein conformational transition and dynamics within adsorbent pore due to the lack of microscopic experimental techniques. In the present study, a coarse-grained adsorbent pore model established in an earlier work is used to represent the actual porous adsorbent composed of matrix and immobilized HCIC ligands. Two adsorbent pores with different ligand distributions are constructed by adjusting the coupling sites, denoted as L1 and L2. In L1 the ligands are bonded uniformly while in L2 the ligands are arranged in lines in the axial direction and thus exhibit a heterogeneous distribution. Protein adsorption, desorption, and conformational transition in both L1 and L2 are shown by molecular dynamics simulations of a 46-bead beta-barrel coarse-grained model protein within the adsorbent pore models. The simulations indicate that ligand homogeneity has significant effect on both the irreversibility and the dynamics of adsorption while no obvious effect on protein conformation distribution. In comparison with L1, L2 leads to irreversible and slow adsorption, indicating the strict requirement of a suitable protein orientation to reach stable adsorption. The simulations have provided new insight into the microscopic behavior of HCIC, which would be beneficial to the rational design of adsorbents and parameter optimization for high-performance HCIC., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Electro-kinetic acceleration of protein mass transfer was studied by dynamic chromatographic experiments and computer simulations on IEC with an oscillatory transverse electric field (EF) perpendicular to the mobile-phase flow (pIEEC). The breakthrough behavior of BSA was investigated at different electric current densities. A two-dimensional mathematical model has been established to describe the electro-kinetic convection-diffusion behavior of protein adsorption in a single adsorbent particle. The equation was coupled with the axial dispersion model to simulate the dynamic adsorption process in the pIEEC. The model parameters were determined by independent experiments or calculations. It was found that protein adsorption led to an exponential decrease of intraparticle electro-kinetic flow with increasing protein adsorption. Taking this effect into consideration, the model calculations could well describe dynamic breakthrough curves. Moreover, protein distribution in adsorbents was observed to present excursion along the periodical oscillatory EF direction. At the beginning of pIEEC, intraparticle convection caused by the EF contributed more to the enhancement of dynamic binding capacity. Finally, it was confirmed that both the EOF and electrophoresis contributed to the acceleration of mass transfer.

High ligand density is usually required in hydrophobic charge induction chromatography (HCIC) for high adsorption capacity. However, it is not clear to what extent the ligand density alters the adsorption and desorption behaviors, or if this leads to the protein conformational transition within adsorbent pores. In the present study, molecular dynamics simulation is performed to examine the effects of ligand density in HCIC using a 46-bead beta-barrel coarse-grained model protein and a coarse-grained adsorbent pore model established in our earlier work. Four ligand densities (1.474, 1.769, 2.212, and 2.949 micromol/m(2)) are simulated at 298.15 K. The simulations indicate that both the capacity and irreversibility of adsorption increase with ligand density. However, it is found that the fastest adsorption occurs at a ligand density of 2.212 micromol/m(2) rather than at the highest density studied. Analyses of adsorption trajectories, protein-ligand interaction energy, and the free energy map indicate that there is repulsion of protein when unfavorable contacts of the protein and ligands occur. There is an enhanced repulsion at 2.949 micromol/m(2), which increases the energy barrier to the transition region and reduces the opportunities to get stable adsorption, thus leading to the decreased adsorption rate. At 2.212 micromol/m(2), however, the repulsion is mild and the high ligand coverage provides abundant opportunities for the protein to get the fastest adsorption and thus causes the maximum unfolding. In the following simulations, complete and irreversible desorption is observed at all ligand densities, in agreement with the easy pH-induced elution behavior of HCIC observed experimentally. It is found that there is a suitable balance between hydrophobic attraction and electrostatic repulsion at 2.212 micromol/m(2), which leads to the slowest desorption kinetics and causes the maximum unfolding. Moreover, analysis of unfolded protein distribution indicates that unfolding occurs mainly on the ligand surface in both adsorption and desorption. The behaviors have been comprehensively elucidated by molecular and thermodynamic analyses.

Aim: The present study aims to investigate the effect of nourishing "Yin"-removing "Fire" herbal mixture, a Chinese herb-based formulation, on hypothalamic kisspeptin expression in danazol-induced female precocious model rats., Materials and Methods: The female Sprague-Dawley rats were divided into intact normal (N), central precocious puberty (CPP) model (M), vehicle without CPP (V), CPP model exposed to herbal mixture (HM) and CPP model exposed to saline (S) groups. At postnatal day 5, a single subcutaneous injection of 300 microg of danazol was administered to induce CPP model rats. From P15, rats in the HM group were continuously gavaged with the 1 ml/50 g body weight mixture, until two consecutive regular estrous cycles were established. The hypothalamic Kiss-1 expression was detected by RT-PCR and immunohistochemistry., Results: The day of vaginal opening and establishment of two regular estrous cycles were delayed in the HM group compared with M and S groups (P<0.05, respectively). The level of hypothalamic Kiss-1 mRNA and the number of kisspeptin-immunoreactive (kisspeptin-ir) cells in the arcuate nucleus (ARC), preoptic area (POA) and periventricular nucleus (PeN), were decreased significantly in the HM group compared with the M and S groups (P<0.01, respectively) on the day of onset-puberty., Conclusion: These results indicate that the kisspeptin signaling pathway might be involved in the effect of herbal mixture treatment on CPP., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Mixed-mode chromatography is a chromatographic method that utilizes more than one form of interactions between the stationary phase and the solutes in a feed stream. Compared with other types of chromatography, mixed-mode chromatography is advantageous in its salt-independent adsorption, facile elution by charge repulsion, and unique selectivity. Hence, it has already proved beneficial for the separation of proteins as well as other purposes. In this article, mixed-mode ligands for protein purification have been reviewed. These ligands usually have an aliphatic or aromatic group as the hydrophobic moiety and an amino, carboxyl or sulfonic group as the ionic moiety. Heterocyclic groups are good ligand candidates for their unique hydrophobicity and dissociation property. Hydrogen bonding groups also have influences on the performance of mixed-mode adsorbents. These principles should be considered in the screening and design of mixed-mode ligands. Strategies for the design of synthetic affinity ligands, especially the bioinformatics and combinatorial methods, may be adopted for mixed-mode ligand design. More efforts are needed for the development of rational design and screening methods for mixed-mode protein ligands by sophisticated computational and experimental approaches.

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.

Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method has been successfully applied in the separation and purification of antibodies and other proteins. However, little is understood about protein conformational transition and the dynamic process within adsorbent pores. In the present study, a pore model is established to represent the realistic porous adsorbent composed of matrix and immobilized HCIC ligands. Protein adsorption, desorption, and conformational transition in the HCIC pore and its implications to the separation performance are shown by a molecular dynamics simulation of a 46-bead beta-barrel coarse-grained model protein in the adsorbent pore. Repeated adjustment of both protein position and orientation is observed before reaching a stable adsorption. Once the protein is adsorbed, there is a dynamic equilibrium between unfolding and refolding. The effect of hydrophobic interaction strength between protein and ligands on adsorption phenomena is then examined. Strong hydrophobic interaction, representing the presence of high-concentration lyotropic salt in mobile phase, can speed up the adsorption but cause protein unfolding more significantly. On the contrary, weak hydrophobic interaction, representing the absence of a lyotropic salt or the presence of a chaotropic agent, can reserve native protein conformation but does not lead to stable adsorption. In the elution, protein unfolding occurs due to simultaneous hydrophobic adsorption and electrostatic repulsion in the opposite directions. When the protein has been desorbed, the conformational transition between unfolded and native protein is still observed due to the long-range nature of electrostatic interaction. The simulation has provided molecular insight into protein conformational transition in the whole HCIC process, and it would be beneficial to the rational design of ligands and parameter optimizations for high-performance HCIC.

A novel negatively charged tentacle-type polymer-coated capillary column was fabricated and applied for on-line extraction and preconcentration of proteins. The polymer coating was prepared by glycidyl-methacrylate graft polymerization in a silanized capillary column and the following sulfonic acid group functionalization. It had high surface area and offered high phase ratio for protein adsorption. In addition, the polymer-coated capillary column provided more stable EOF than a bare uncoated capillary. These features of the polymer coating facilitated the extraction of proteins through electrostatic interactions. This was used to extract proteins. The extracted analytes were then desorbed and focused by EOF in the direction opposite to the sample injection flow for subsequent CE. With this procedure, over 1500-fold sensitivity enhancement was realized for myoglobin (MB) as compared with a normal capillary zone electrophoresis. By comparison of the peak areas of the enriched protein, it was found that the polymer-coated column could capture proteins about 30 times more than the uncoated column. In addition, the separation of a protein mixture containing 0.4 microg/mL of MB and 0.4 microg/mL of insulin was demonstrated by the on-line preconcentration and electrophoretic separation with the polymer-coated column.

The use of a phenylalanine (Phe) functionalized tentacle-type polymer coated capillary column for protein separation by open tubular capillary electrochromatography (OTCEC) was demonstrated in this work. The tentacle-type stationary phase was prepared from silanized fused-silica capillaries of 50 microm I.D. by glycidyl methacrylate graft polymerization and subsequent Phe functionalization. Due to the amphoteric functional groups of the Phe bonded on the tentacle-type polymer stationary phase, protein separation in the prepared column can be performed under both cathodic and anodic electroosmotic flow (EOF) by varying the pH values of the mobile phase. Model proteins including ribonuclease A (RNase A), myoglobin, transferrin, insulin were baseline separated under cathodic EOF with a mobile phase of pH 8.8. Comparison between the separation result of the four proteins under conditions of OTCEC and capillary zone electrophoresis indicates that the migration behavior of the four proteins in the prepared column was the result of the interplay of chromatographic retention and electrophoretic migration. Besides, three basic proteins including RNase A, cytochrome c (Cyt-c) and lysozyme (Lys) were fully resolved under anodic EOF with an acidic running buffer (pH 2.5). The elution order was the same as the isoelectric point values of the proteins (RNase A

A novel approach for the fabrication of macroporous poly(glycidyl methacrylate-ethylene glycol dimethacrylate) monolith is presented. The method involved the use of sodium sulfate granules and organic solvents as co-porogens. Compared with the conventional monoliths [ML-(1-3)] using organic solvents only as a porogen, the improved monoliths [MLS-(1-3)] showed not only higher column efficiency and dynamic binding capacity (DBC) for protein (bovine serum albumin, BSA), but also higher column permeability and lower back pressure. It is considered that the superpores introduced by the solid granules played an important role for the improvement of the monolith performance. Moreover, poly(glycidyl methacrylate-diethylamine) tentacles were grafted onto the pore surface of MLS-3 monolith. This has further increased the DBC of BSA to 74.7 mg/ml, about three times higher than that of the monoliths without the grafted tentacles. This grafting does not obviously decrease the column permeability, so a new monolith of high column permeability and binding capacity has been produced for high-performance preparative protein chromatography.

To investigate the potential role of kisspeptin in the advance onset of puberty in precocious puberty, model rats induced by danazol were used to study the developmental expression of hypothalamic kisspeptin. Kisspeptin immunoreactive cells were observed in the arcuate nucleus (ARC), periventricular nucleus (PeN) and preoptic area (POA) in model rats on the day of onset-puberty. On the day of post-puberty, however, the number of kisspeptin immunoreactive cells in ARC and PeN decreased while the number of those cells in POA increased. Kisspeptin immunoreactive cells were not detected in hypothalamus in both normal and model rats at their pre-puberty stages. Furthermore, the expression of hypothalamic Kiss-1 mRNA reached top on the day of onset-puberty in both of the normal and model rats, and the expression of Kiss-1 mRNA increased significantly in the model rats compared with those in the normal ones. Our results indicated that kisspeptin might involve in the advance onset of puberty in danazol induced female precocious model rats.

This article describes the fabrication of a rigid magnetic monodisperse bead (M-PGMA-TRI, 4.92 microm) with polyglycidyl methacrylate (PGMA) cross-linked by trimethylolpropane trimethacrylate (TRI). This was realized by adding a proper amount (2%, w/w) of TRI after 3 h of the dispersion-polymerization reaction with the monomer of GMA. The mono-sized microspheres were further processed to introduce magnetic granules by sulfonation and penetration-deposition approaches. The monodisperse bead (M-PGMA) without TRI addition was also fabricated for comparison. The morphology, size and magnetic characteristics of the microspheres were extensively characterized. The M-PGMA-TRI microspheres were nonporous, of smooth surface and superparamagnetic with a saturation magnetization of 13.0 emicro/g. Recycled use of the material for protein adsorption exhibited stability of the magnetic properties of the M-PGMA-TRI, as compared to the significant loss of the saturation magnetization of the M-PGMA. The chemical stability of the M-PGMA-TRI was also confirmed by examining its protein adsorption and magnetic properties after incubation in various solutions such as acidic buffer (pH 2.2) for 24 h. The adsorption capacity of gamma-globulin reached 287.2 mg/g and kept stable in the repeated adsorption/desorption/regeneration cycles. The results indicated that the introduction of 2% TRI was promising for producing rigid magnetic mono-sized microspheres for protein adsorption.

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigenesis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.

Rational design of affinity peptide ligands of proteins by flexible docking simulation is performed using the SYBYL program package. This approach involves the use of experimental data to verify a scoring function that can be used to assess the affinity of a peptide for its target protein. The enzyme-linked immunosorbent assay (ELISA) data of several peptides displayed on phage surfaces for insulin and lysozyme, respectively, reported in literature are used for the purpose. It is found that the absolute values of the Dscore calculated from the docking correspond well to the ELISA data that relate to the affinity between the peptides and the target molecule. So, the Dscore function is used to assess the affinity of docked peptides in a pentapeptide library designed on the basis of protein (alpha-amylase) structure. As a result, a pentapeptide with a high Dscore value is selected and a hexapeptide (FHENWS) is built by linking serine to its C-terminal to lengthen the peptide. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions (including hydrogen bonds) and Van der Waals forces contribute to the affinity of the hexapeptide for alpha-amylase. Chromatographic experiments with the immobilized peptide have given further evidence for this observation. Adsorption isotherm described by the Langmuir equation indicates that the apparent binding constant of alpha-amylase to the immobilized hexapeptide was 2.5x10(5)L/mol. Finally, high affinity and specificity of the affinity adsorbent is exemplified by the purification of alpha-amylase from crude fermentation broth of Bacillus subtilis.

Background: Advanced oxidation protein products (AOPPs) are new uremic toxins reported by Witko-Sarsat in 1996, which are associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of AOPPs on MCP-1 expression in cultured vascular smooth muscle cells (VSMCs)., Methods: VSMCs were cultured and then co-incubated with AOPP (200 micromol/L, 400 micromol/L) for different times with or without pretreatment with specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. RT-PCR and Western blott were used to detect MCP-1 mRNA and protein expression at different time points after AOPP stimulation in rat smooth muscle cells. Western blot was used to detect the expression of phosphorylated p38 MAPK., Results: Treatment of VSMC with AOPPs resulted in a significant increase of the expression of MCP-1 mRNA and protein in time- and dose-dependent manner, and could activated p38 MAPK. Pretreatment of VSMCs with SB203580 resulted in a dose-dependent inhibition of AOPPs-induced MCP-1 mRNA and protein expression., Conclusions: AOPPs can stimulate MCP-1 expression via p38 MAPK in VSMCs. This suggests that AOPPs might contribute to the formation of atherosclerosis through this proinflammatory effect.

The mass transfer of bovine serum albumin (BSA) to a cation exchanger, SP Sepharose FF, has been studied by finite batch adsorption experiments. The uptake curve was simulated with three mass transport models (i.e., effective pore diffusion model, surface diffusion model and Maxwell-Stefan model) incorporating the particle size distribution of the adsorbent particles. All the three models can simulate the uptake curves reasonably well. However, how well these models could simulate the real concentration profile within the adsorbent particle cannot be verified by the fitness of the models to the uptake curve. Thus, confocal laser scanning microscopy (CLSM) was used to visualize protein uptake to the porous adsorbent particles during the batch experiments. Using a fluorescent dye-labeled bovine serum albumin (BSA) for the dynamic adsorption experiments, the radial concentration profiles of the labeled BSA molecules into individual adsorbent particles at different times were obtained from the CLSM images. The protein distribution profiles within various particle diameters at different time were compared with the radial protein distributions predicted from the models. It reveals that surface diffusion model describes the intraparticle protein concentration profiles better than the other two models.

Ion-exchange electrochromatography with an oscillatory electric field perpendicular to mobile-phase flow driven by pressure (pIEEC) was developed with a column design of rectangle cross-section. The effect of electric field strength on the dynamic binding capacity (DBC) was examined by frontal analysis of bovine serum albumin (BSA) adsorption to the packed beds of DEAE Sepharose FF in Tris-glycine buffer (pH 8.2). It was shown that the DBC at 10% breakthrough (Q(10)) in the pIEEC increased linearly with increasing the electric field strength. For example, with a packed-bed height of 15mm and electric potential gradient of 38V/cm, Q(10) increased four times over that in normal ion-exchange chromatography. So, the transverse electric field has created significant electro-kinetic mass transports (electroosmosis and electrophoresis) that intensified exterior liquid-film and intraparticle mass transfers, leading to the increased protein binding capacity. Due to the increased capacity in the pIEEC, partial resolution of BSA and IgG under an overload condition was realized without any process optimization. The results have revealed that an electric potential gradient of 20V/cm was enough to greatly enhance the DBC in the pIEEC, and when necessary, high electric field strength can be realized with a low applied voltage because the side distance of the column is usually an order of magnitude smaller than its height. The use of low voltage to carry out electrochromatography is a significant advantage of the pIEEC over conventional electrochromatography with axial electric field.

A novel superporous agarose (SA) bead characterized by the presence of wide pores has been fabricated by water-in-oil emulsification using solid granules of calcium carbonate as porogenic agent. After cross-linking, the solid granules were removed by dissolving them in hydrochloric acid. Then, the gel was modified with diethylaminoethyl groups to create an anion exchanger, SA-DEAE, for protein adsorption. A homogeneous agarose (HA) bead was also produced and modified with DEAE for comparison. It was found that the porosity of SA-DEAE was about 6% larger than that of HA-DEAE. Moreover, both optical micrographs and confocal laser scanning microscopy (CLSM) of the ion exchangers with adsorbed fluorescein isothiocyanate (FITC) labeled IgG revealed the superporous structure of the SA medium. In addition, the SA-DEAE column had lower backpressure than the HA-DEAE column, confirming the convective flow of mobile phase through the wide pores. Due to the presence of the wide pores, more channels were available for protein transport and, furthermore, more diffusive pores in the agarose network were accessible for the protein approach from different directions. This led to 40% higher protein capacity and two times higher effective pore diffusivity in the SA-DEAE than in HA-DEAE. Moreover, an increase of the efficiency of the SA-DEAE column until a flow rate of 5 cm/min and the independency of the column efficiency at flow rates from 5 to 17.8 cm/min was found, indicating that intraparticle mass transfer was intensified by convective flow at elevated flow rates. Therefore, the chromatographic resolution of IgG and BSA was little affected up to a flow rate of 17.8 cm/min. The results indicate that the SA medium is favorable for high-speed protein chromatography., ((c) 2005 Wiley Periodicals, Inc.)

A novel preparative size-exclusion electrochromatography with an oscillatory low-voltage electric field perpendicular to the liquid phase streamline (pSEEC) was proposed with a column design of rectangular cross-section. The column of 12 cm length was packed with Sephadex G-75, and the retention behavior of bovine serum albumin (BSA) and myoglobin (Myo) was extensively investigated under various conditions. The results indicated that the partition coefficient of a charged protein increased significantly on increasing the current strength as well as the difference between its pI and pH. The partition coefficient also increased on decreasing the mobile phase conductivity. For the gel-excluded protein like BSA, the concentration polarization (CP) on the gel surface induced by the protein electromigration was the main reason for the increased retention. For a gel-permeable protein like Myo, both the CP and electrophoretic migration in the solid phase contributed to its increased retention. Further results exhibited that the polarization would be offset by diffusion, because the accumulated protein would flux back to the bulk liquid phase. Therefore, when the electrically induced mass flux was equal to the diffusion flux, the partition coefficient did not change with a further increase of the oscillatory current cycle. Finally, pSEEC was compared with SEC in the separation of protein mixtures of BSA/Myo as well as BSA/Myo/lysozyme. The results showed much better resolutions of the protein mixtures in pSEEC with the column as short as 12 cm. The potential of pSEEC for preparative protein separation was therefore demonstrated.

A novel rigid biporous bead (BiPB) had been fabricated by double emulsification to prepare a (w/o)/w emulsion and a subsequent polymerization. The polymerization of monomers, glycidyl methacrylate and ethylene glycol dimethacrylate, was initiated with benzoin ethyl ether by ultraviolet irradiation. The BiPB with an average diameter of 42.8 microm was characterized to possess two types of pores, i.e., micropores (20-100nm) and superpores (300-4000nm). Its specific surface area was determined to be 41.9m2/g, about 20% smaller than that of a microporous bead (MiPB) (52.1 m2/g). Flow hydrodynamic experiments showed that the BiPB column had smaller backpressure and plate height than those of the MiPB column at a given flow rate. Derivatized with diethylamine (DEA), the static adsorption capacity of the DEA-BiPB was about 7% smaller than that of the DEA-MiPB for BSA (bovine serum albumin). However, frontal analysis demonstrated that the dynamic binding capacity of the DEA-BiPB column was 1.6-2.4 times higher than that of the DEA-MiPB at high flow rate range of 1200-2400cm/h. Moreover, separation of a model protein mixture (myoglobin and BSA) was conducted at mobile phase velocities up to 3000cm/h to compare the performance of the two stationary phases. All the results indicate that the BiPB contains interconnected flowthrough pores and the BiPB column is promising for high-speed protein chromatography.

Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first native-like pore-forming protein, small globular protein (SGP). We show here that this artificially engineered protein has membrane-disrupting properties and anti-tumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications.

Development of a high-performance stationary phase is an essential demand for high-speed separation of proteins by liquid chromatography. Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and a mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores, that is, micropores (10-90 nm) and macropores (180-4000 nm). Furthermore, the biporous medium possesses specific surface area as high as 91.3 m(2)/g. Because of the presence of the macropores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was found to be as high as 54.6 mg/g wet bead at 300 cm/h, approximately 63.2% of its static capacity. In addition, the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000 cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.