In this study, a completely green and facile protocol to oriented immobilization of histidine-tagged (His-tagged) proteins based on plant polyphenolic tannic acid (TA) is described. This is the first time that TA has been applied as ionic chelators to immobilize His-tagged proteins. To reduce the nonspecific interactions between the TA and immobilized proteins, we assembled nonfouling zwitterionic poly(sulfobetaine methacrylate) (PSBMA) on the TA surface. The use of PSBMA could maintain the high activity of the His-tagged proteins and inhibit the adsorption of untagged protein to the TA surface. Subsequently, the obtained TA/PSBMA film was further chelated with CoII for specific binding to a His-tagged protein. As CoIII is more stable and inert than CoII, the chelated CoII was oxidized to CoIII. Using this approach, His-tagged Chitinase was anchored to TA/PSBMA/CoIII film as a catalyst for the hydrolysis of chitin. The loading capacity of the film for the His-tagged Chitinase can reach ∼4.0 μg/cm2. Moreover, the oriented immobilized Chitinase had high catalytic activity and excellent thermal and storage stability as well as being more resistant to proteolytic digestion by papain. This low-cost and green protein-oriented immobilization strategy may serve as a versatile platform for a range of applications, such as biomaterials, biocatalysis, sensors, drug delivery, and so on.Keywords: cobalt; His-tagged proteins; nonfouling; oriented immobilization; tannic acid;

Fibrinogen (Fgn) has been identified as the key protein in the process of biomaterial-induced platelet adhesion. We have recently reported a facile and effective method for constructing platelet-repellent surface using a natural polyphenol component tannic acid (TA). However, the mechanism by which the TA surface repels platelets was not fully understood. To address this issue, we investigated the adsorption of Fgn (amount and conformation) on four TA-functionalized surfaces with different amounts of galloyl groups and the potential for platelet adherence on these surfaces. The experimental results indicated that the four TA-functionalized surfaces adsorbed a similar amount of Fgn, but the conformation and bioactivity of the adsorbed Fgn and the subsequent platelet adherence were quite different among the surfaces. The TA surface with the most galloyl groups induced minimal changes in the conformation of Fgn, a result of the α and γ chains of the adsorbed Fgn being highly inactive on the surface, thus leading to an outstanding antiplatelet adhesion performance. With a decreased amount of galloyl groups, the activity of the α chain in the adsorbed Fgn remained unchanged, but the activity of the γ chain and the extent of platelet adhesion gradually increased. This work provided a new concept for controlling platelet adhesion on solid materials, and we envision that the TA film could have potential applications in the development of new blood-contacting biomaterials in the future.Statement of SignificanceReducing platelet adhesion on material surfaces is of tremendous scientific interest in the field of blood-contacting biomaterials, but it remains a big challenge due to the highly adhesive nature of the platelets. In this study, we demonstrated for the first time that tannic acid surface with abundant galloyl groups could induce minimal conformational changes of fibrinogen, eventually leading to an outstanding antiplatelet adhesion effect. In addition, the platelet adhesion response could be easily controlled through regulating the amount of galloyl groups on the surface. This work provided a new strategy for controlling platelet adhesion on solid materials, which was totally different from existing methods such as construction of physically patterned surfaces, modification of inert hydrophilic polymers or appending bioactive moieties to target surfaces.Download high-res image (183KB)Download full-size image

Surface patterning provides a powerful tool to the diagnosis of platelet adhesion. However, the current methodologies of constructing platelet-patterned surfaces require laborious and complicated steps. Herein, a novel and simple platelet-repellent surface was reported by metal (Fe3+ ions)–polyphenol (tannic acid, TA) coordination interaction. The platelet-repellent effect was significantly better than that of poly(ethylene glycol) (PEG) in a long-term. Moreover, the platelet-repellent behavior could extend to other polyphenols-functionalized surfaces. On the basis of these observations, a TA-based micropattern was fabricated in situ by one-step microcontact printing for well-defined platelet adhesion, which can effectively avoid the traditional introduction of inert hydrophilic polymers and bioactive ligands. Afterward, the TA-based micropattern was applied to monitor the adhesion of defective platelets treated with an antiplatelet drug (tirofiban). This work provided a facile, versatile, and environmentally friendly strategy to construct platelet-repellent polyphenolic surfaces and their micropattern. We expect that this simple micropattern could act as a low-cost and label-free platform for biomaterials and biosensors, and could be widely used in the clinical diagnoses of platelet adhesive functions and the evaluation of antiplatelet therapies.Keywords: antiplatelet drug; micropattern; platelet adhesion detection; platelet-repellent surfaces; polyphenols

A strategy for photoinduced covalent immobilization of proteins on phenol-functionalized surfaces is described. Under visible light irradiation, the reaction can be completed within seconds at ambient temperature, with high yields in aqueous solution of physiological conditions. Protein immobilization is based on a ruthenium-catalyzed radical cross-linking reaction between proteins and phenol-modified surfaces, and the process has proven mild enough for lipase, Staphylococcus aureus protein A, and streptavidin to preserve their bioactivity. This strategy was successfully applied to antibody immobilization on different material platforms, including agarose beads, cellulose membranes, and glass wafers, thus providing a generic procedure for rapid biomodification of surfaces.

DDT (1,1,1-trichloro-2.2-bis(p-chlorophenyl)ethane), one of the most abused insecticides, is a highly hazardous component for both human health and environmental applications. The biodegradation of DDT into non-toxic, environmentally benign components is strongly limited by the poor bioavailability of DDT. In this work, we combined experiments and molecular simulations to examine the effect of three cyclodextrins (α-, β-, and γ-CD) on their structure-specific interactions with DDT, specifically in relation to DDT solubility and biodegradability. It was found that all three CDs were able to bind to DDT with their inner hydrophobic cavity and different binding affinities and orientations, demonstrating their ability to improve DDT solubility. Different from the strong binding between DDT and β-/γ-CDs via a fully DDT bury mode, α-CD had a relatively weak binding with DDT via a partial DDT bury mode, which allowed DDT to be readily disassociated from α-CD at the lipid membrane interface, followed by DDT permeation into and across the cell membrane. The different binding modes between DDT and CDs explain why only α-CD can promote the bioavailability and biodegradation of DDT by simultaneously increasing its aqueous solubility and membrane interaction. This work provides structural-based binding information for the further modification and optimization of these three CDs to enhance their solubility and biodegradability of DDT.

Human serum albumin (HSA) is an important binding partner of amyloid-β (Aβ) in vivo and it can modulate Aβ aggregation. However, the underlying molecular mechanism of this HSA-mediated modulation of Aβ aggregation and cytotoxicity is still not fully understood, especially that of Aβ42, which is the most amyloidogenic and toxic Aβ variant. For this reason, we systematically investigated the effect of HSA on the fibrillation and cytotoxicity of different Aβ42 aggregation species in the amyloid-formation pathways by extensive biophysical and biological tests. Moreover, a Surface Plasmon Resonance (SPR) assay was performed to determine the ability of HSA to bind to different Aβ42 species. Collective results indicated several important findings as follows: (i) HSA inhibited the fibrillation of the Aβ42 monomer in a concentration-dependent manner; (ii) HSA abolished the seeding ability of protofibril and fibril at a 1:1 molar ratio; (iii) HSA interacted with Aβ42 protofibrils and fibrils with increased affinity and formed HSA–Aβ complexes that dissociated at a slower rate than the complex formed between HSA and the Aβ42 monomer; (iv) HSA prevented seeding-mediated cytotoxicity of Aβ42. Taken together, these findings suggested that the HSA inhibited Aβ42 fibrillation and cytotoxicity through interfering with different stages of Aβ42 fibrillation and targeting different Aβ42 intermediate aggregates. Furthermore, HSA preferentially interacted with Aβ fibrillar aggregates to form slowly-dissociated complexes. These findings contributed to a better understanding of the molecular mechanism by which HSA modulates the aggregation and cytotoxicity of Aβ, and provide important implications for further designing HSA-based therapeutic strategies.

•A site-specific coupling method was applied for oriented immobilization of bioaffinity ligands.•Site-specific aldehyde-modification was achieved by formylglycine-generating enzyme (FGE) in vivo.•The chemical selectivity could enable the immobilization of aldehyde-modified protein A in crude E. coli cell extract without purification.Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli. The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100 mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications.

As a pathogenic toxin, bilirubin is generally removed from blood by hemoperfusion for the remission of liver disease or to gain time for patients waiting for liver transplantation. However, the development of bilirubin adsorbents with excellent mechanical properties, adsorption performance and hemocompatibility is still a considerable challenge. In this work, a heparin-modified chitosan/graphene oxide hybrid hydrogel (hep-CS/GH) has been developed for bilirubin adsorption using a lyophilization–neutralization–modification strategy. The as-prepared hybrid hydrogel displayed a unique foam-like porous structure and excellent mechanical flexibility. It was revealed that the incorporation of graphene oxide into the chitosan matrix enhanced both the compressive strength and the Young's modulus of the hybrid hydrogel, as well as its adsorption capacity for bilirubin. The maximum adsorption capacity of hep-CS/GH for bilirubin was 92.59 mg g−1, according to the Langmuir isotherm model. It was demonstrated that hep-CS/GH successfully competed with albumin, and could effectively adsorb bilirubin from a bilirubin-enriched serum. After the hydrogel was modified with heparin, protein adsorption, platelet adhesion and hemolysis were reduced, and the plasma clotting time was prolonged from 4.1 to 23.6 min, indicating the superior hemocompatibility of hep-CS/GH. Therefore, this study may pave the way for improving the performance of the adsorbent in removing blood toxins.

Amyloid-β (Aβ) fibrillation is a crucial factor in the etiology of Alzheimer's disease (AD). Curcumin has been widely studied and considered as a promising drug for AD treatment, but its molecular mechanism in inhibiting Aβ aggregation is still not clearly defined. In the present study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to analyse the growth behaviors of Aβ fibrils in the presence and absence of curcumin. The viscoelasticity properties that reflect the structural information of the resulting Aβ aggregates were also analysed using a ΔD/ΔF plot. It was found that the presence of curcumin could accelerate the deposition process of Aβ monomers on the initially immobilized fibrils, with the growth rate being 21.1–120.6% higher than that of the control. Viscoelasticity analysis showed that curcumin-induced Aβ aggregates exhibited a much more flexible structure than that of the initial fibrils, and the degree of this kind of structural conversion was related to the concentration of curcumin. Importantly, we found that further deposition of Aβ monomers on these loosely constructed Aβ aggregates was significantly inhibited, with the growth rate being only 34.2% of initial rate. Therefore, the QCM-D study demonstrated that curcumin inhibited the growth of Aβ fibrils through a process that led to the structural conversion of the growing fibrils, and directed these fibrils to an off-pathway aggregation, which may hinder the formation of long Aβ fibrils that could cause neuronal damage.

Unwanted adhesion of microalgae on submerged surfaces is a ubiquitous problem across many maritime operations. We explored the strategy of developing a silver nanoparticle (AgNP) coating for antifouling applications in marine and freshwater environments. In situ growth of AgNPs was achieved by a polydopamine (PDA)-based method. A range of most used industrial materials, including glass, polystyrene, stainless steel, paint surface, and even cobblestone, were employed, on which AgNP coatings were built and characterized. We described the fouling-resistant behavior of these AgNP-modified surfaces against two typical fouling organisms: a marine microalga Dunaliella tertiolecta and a freshwater green alga community. The PDA-mediated AgNP deposition strategy was demonstrated applicable for all the above materials; the resulting AgNP coatings showed a significant surface inhibitory effect against the adhesion of microalgae by above 85% in both seawater and freshwater environments. We observed that contact killing was the predominant antifouling mechanism of AgNP-modified surfaces, and the viability of the microalgae cells in bulk media would not be affected. In addition, silver loss from PDA-mediated AgNPs was relatively slow; it could allow the coating to persist for long-term usage. This study showed the potential of preparing environmentally friendly surfaces that can effectively manage biofouling through the direct deposition of AgNP coatings.Keywords: antifouling coating; Dunaliella tertiolecta; marine fouling; polydopamine; silver nanoparticle;

•Competitive elution for HCIC is based on MEP/β-CD supermolecular interaction.•MEP/β-CD inclusion complex relies on hydrophobic interaction and H-bond.•15 mM β-CD allows IgG elution from HCIC at neutral pH.Hydrophobic charge-induction chromatography (HCIC) has emerged as a useful addition to Protein A chromatography for antibody purification due to its remarkable merits in cost and stability. However, the instability of antibody during acidic elution, which may cause inactivation and aggregation, is still a major concern for the efficiency of this method. The aim of this study is to develop a new strategy of competitive elution with inclusion complexes in HCIC, and to apply it to antibody elution under neutral pH conditions. Interactions between 4-mercaptoethylpyridine (MEP), a typical ligand of HCIC, and four different types of cyclodextrins (CDs) were investigated by molecular docking; immunoglobulin G (IgG) elution capacities of CDs were characterized on MEP-based HCIC mediums. The results demonstrated the general effectiveness of CD-based eluents for HCIC. This type of displacement eluents could allow an efficient elution of bound antibody over a broad range of pH and ion strength. With 15 mM β-CD, elution of human IgG was achieved at physiological pH, with an average IgG recovery of 87%. When this elution strategy was used to separate antibody directly from human serum, substantial elution of bound IgG could be obtained at pH 7.4, with product purity comparable to traditional method with an acidic buffer. We expect such method can be of special interest in developing HCIC elution strategy for the proteins like antibody that are sensitive to acidic conditions.

•Hydrophobicity-intensified HIC resins can allow the capture of antibody at the physiological salt concentration.•Displacement elution of HIC can be achieved through phenyl/β-CD supermolecular interaction.•β-CD has an association constant (Ka) of 4.1 × 103 M−1 with the ligand.•Salt-independent hydrophobic displacement chromatography has proven effective in direct purification of IgG from human serum.Hydrophobic interaction chromatography (HIC) offers an orthogonal selectivity to ion exchange chromatography and the combination of the two processes can provide a potential cost-effective alternative to protein A chromatography in industrial antibody purification. However, the application of HIC is limited by its close dependence on high concentrations of kosmotropic salts to achieve desired separation. These salts can cause antibody precipitation and induce the corrosion of manufacturing facilities. Here, we report a new strategy of salt-independent HIC, which can capture antibody at the physiological salt concentration and allow the recovery of bound proteins through cyclodextrin (CD)-based displacement elution. Hydrophobicity-intensified HIC media with different coupling amount of phenyl ligands were prepared and assessed for their antibody binding capacity and selectivity. β-CD was investigated for its supermolecular interaction with phenyl ligands and elution capacity as a displacer. The results clarified a nearly linear correlation between binding capacity of human immunoglobulin G (IgG) and phenyl coupling density in the range of 44–159 μmol/mL. The host–guest interaction between β-CD and the phenyl ligands revealed a modest binding strength (Ka = 4.1 × 103 M−1), and 15 mM β-CD solution showed a general effectiveness as displacement eluent for these HIC media, with IgG recovery varying with the ligand density. This strategy allowed the direct purification of human IgG from serum with satisfactory purity. The whole procedure of this method, including loading and elution, can be performed under physiological conditions. We expect such a salt-independent mode of HIC could be used as a capture or intermediate step in industrial antibody purification.

Optical biosensing techniques have become of key importance for label-free monitoring of biomolecular interactions in the current proteomics era. Together with an increasing emphasis on high-throughput applications in functional proteomics and drug discovery, there has been demand for facile and generally applicable methods for the immobilization of a wide range of receptor proteins. Here, we developed a polymer platform for microring resonator biosensors, which allows the immobilization of receptor proteins on the surface of waveguide directly without any additional modification. A sol–gel process based on a mixture of three precursors was employed to prepare a liquid hybrid polysiloxane, which was photopatternable for the photocuring process and UV imprint. Waveguide films were prepared on silicon substrates by spin coating and characterized by atomic force microscopy for roughness, and protein adsorption. The results showed that the surface of the polymer film was smooth (rms = 0.658 nm), and exhibited a moderate hydrophobicity with the water contact angle of 97°. Such a hydrophobic extent could provide a necessary binding strength for stable immobilization of proteins on the material surface in various sensing conditions. Biological activity of the immobilized Staphylococcal protein A and its corresponding biosensing performance were demonstrated by its specific recognition of human Immunoglobulin G. This study showed the potential of preparing dense, homogeneous, specific, and stable biosensing surfaces by immobilizing receptor proteins on polymer-based optical devices through the direct physical adsorption method. We expect that such polymer waveguide could be of special interest in developing low-cost and robust optical biosensing platform for multidimensional arrays.Keywords: microring resonators; optical biosensor; physical adsorption; protein immobilization; sol−gel polymer; surface modification;

Release of heparin from the surface of biomaterials is a feasible and efficient manner for preventing blood coagulation because of the high bioactivity of free heparin and a low application dosage compared to intravenous injection of heparin. Here we report a novel method featuring a blend of heparin-loaded SBA-15, catechol-modified chitosan (CCS), and heparin as a heparin-releasing film. The release of heparin was based on its leakage from heparin-loaded amino-functionalized mesoporous silica SBA-15 (SBA-15-NH2), which was controlled by the amino density of the SBA-15-NH2. Heparin-loaded SBA-15-NH2, CCS, and heparin were mixed together, and the mixture was cast onto the surface of a polydopamine-modified substrate, forming a heparin-releasing film on the surface of the substrate. The polydopamine acted as an adhesive interlayer that stabilized the film coated on the substrate. The sustained release rates of heparin from the film ranged from 15.8 to 2.1 μg/cm2/h within 8 h. The heparin-releasing film showed low fibrinogen adsorption, platelet adhesion, and hemolysis rate, indicating that it has good blood compatibility. This new approach would be very useful for modifying the surface of versatile blood-contacting biomaterials and ultimately improve their anticoagulation performance.Keywords: blood compatibility; heparin; polydopamine; SBA-15; surface modification;

A metal–organic cerium tetrahedron having size constraints and cooperated interactions within its cavity was used to selectively recognize tryptophan over other natural amino acids and Trp-containing peptides. It was applied in quantificational detection of free tryptophan in serum.

Carbon nanotubes (CNTs) have excellent adsorption properties for a wide range of substances, but the leakage of CNTs into blood and the non-specific adsorption of plasma proteins are major issues in the application of CNTs in blood purification. To minimize these shortcomings, multi-walled carbon nanotubes (MWCNTs)/iron oxides magnetic composites were prepared and intended to be used in bound solute dialysis. Magnetic MWCNTs were characterized by FTIR, XRD, SEM, VSM and Boehm titration. Their adsorption characteristic for bilirubin (a prominent marker of liver failure disease) was measured in aqueous solutions. The magnetic MWCNTs that theoretically contained 50% of 4 h-pre-treated MWCNTs and 50% of magnetite could meet the requirements for good dispersion ability, high adsorption capacity for bilirubin, and the convenience of magnetic separation. The magnetic MWCNTs showed a rapid rate for the uptake of bilirubin, and maximum adsorption capacity could reach 263.16 mg/g at 30 °C. Besides, the adsorption isotherm was well described by Langmuir model, and the adsorption process was thermodynamically favorable. The binding constant for the adsorption of bilirubin to magnetic MWCNTs was 1.8 × 105 M−1 at 30 °C. In bound solute dialysis, 35% of bilirubin could be removed from a solution of albumin without significant effect on the concentration of albumin. This study demonstrated that magnetic MWCNTs could offer a new type of adsorbent for removal of albumin bound toxins in bound solute dialysis.Graphical abstractHighlights► Magnetic MWCNTs could be dispersed in aqueous solution and isolated by a magnet. ► Magnetic MWCNTs showed high adsorption capacity and affinity for bilirubin. ► The adsorption of bilirubin to magnetic MWCNTs was thermodynamically spontaneous and followed the Langmuir model. ► Bilirubin could be removed from albumin solution by magnetic MWCNTs suspension in bound solute dialysis.

The relationship between the selectivity of a particular polychlorinated biphenyls (PCBs) congener and its biodegradability under the same concentration, especially by Enterobacter sp. LY402, is less well studied. To measure congener selectivity of Enterobacter sp. LY402, several influencing factors were studied. The results showed LY402 effectively degraded coplanar 3,4,3′,4′-chlorobiphenyl (CB) at a concentration of 0.05 μM, but not 0.5 μM. The degradation rates of 2,4,5,2′,3′-CB and 2,4,5,2′,4′,5′-CB were increased significantly when the sample constituents were changed from 12 to 5 congeners or to one congener. This indicated that bioremediation of individual congener was affected by other congeners present in the mixture. Moreover, for PCBs containing one chlorine on each phenyl ring, the reactivity preference of LY402 was 2,2′-CB ≥ 3,3′-CB ≫ 4,4′-CB. For two ortho chlorines congeners of PCBs, 2,2′-CB was degraded faster than 2,6-CB. Although 2,6-CB and 4,4′-CB were poorly degraded, the addition of one (i.e., 2,4,4′-CB and 2,6,3′-CB) or two more chlorines (i.e., 2,4,2′,4′-CB) on the phenyl ring significantly increased their biodegradability. In addition, comparing the two congeners of ortho-meta-chlorinated biphenyl, 2,3,2′,3′-CB with neighbor meta chlorines was degraded slower than 2,5,2′,5′-CB with interval meta chlorines. All these indicated that the transformation rates of PCBs were not consistent with the number of chlorines, and PCBs containing the same numbers of chlorines but at different positions also resulted in different conversions. In principle, the extents of effect caused by the position of chlorine substituents on the degradation of PCBs by LY402 were ortho- > meta- > para-CB. In conclusion, the congener selectivity of LY402 was determined by many factors, including the composition of the congeners, their concentrations in the mixture and location and number of chlorine substituents on the phenyl rings.

Aptamer (oligonucleotide) with specific binding capacity for target could be obtained via a screening technique called SELEX (systematic evolution of ligands by exponential enrichment). With the development of them for about two decades, aptamers, as an alternative to antibodies, has shown bright prospects in scientific research, clinical diagnosis, and therapy. As the screening technique is the key to getting an aptamer successfully, new approaches and methods such as subtractive SELEX, complex targets SELEX, genomic SELEX, CE SELEX have appeared continually to improve the screening efficiency in recent years. These methods are reviewed in this article.

Extracorporeal immunoadsorption (ECI) therapy using Staphylococcal Protein A columns has proven effective for removing autoantibodies and circulating immune complexes from patients selectively, providing a promising treatment for autoimmune diseases. However, due to the drawbacks of Protein A in terms of cost and stability, the widespread use of Protein A based ECI is limited. In this study, we investigated the feasibility of 4-mercaptoethylpyridine (MEP, MW 139 Da), a simple and inexpensive synthetic compound, as an alternative to Protein A for autoantibody removal therapy. MEP-based adsorbents were prepared by coupling MEP to Sepharose CL-6B. We found that ligand density was an adjustable parameter for the synthesis of adsorbents aiming at different pathogenic factors, depending on the class of antibody. MEP-Sepharose with a ligand density of 98.8 μmol/ml could remove 80% of the anti-double-stranded DNA antibodies from human serum, whereas a ligand density of 64.5 μmol/ml was enough to remove 96% of the rheumatoid factor (RF) in the serum. Moreover, MEP-based adsorbents showed a lower degree of individual differences compared to Protein A-Sepharose. RF removal of 90% was achieved for all 12 serum samples from different individuals. Among the 14 serum samples derived from systemic lupus erythematosus patients, 11 samples had markedly reduced antinuclear antibody titers. In addition, non-specific adsorption of plasma components to MEP-Sepharose was limited, and the binding capacity of the absorbent for IgG was still about 20 mg/ml of gel after 10 cycles. The results indicated that MEP-based adsorbent could offer a new type of adsorber for the treatment of autoimmune diseases.

•SPA was first successfully expressed as a secretory protein in Pichia pastoris.•The yield of secretory SPA was about 8.8 g/L after fermentation optimization.•The pigments in the fermentation supernatant were removed mostly by desalting.•Only two steps were taken to purify the secretory expression SPA.•The secretory expression SPA was identified to perform excellent IgG binding ability.Staphylococcal Protein A (SPA), a cell wall protein of Staphylococcus aureus, is in high demand because of its ability to bind immunoglobulins. Much of the SPA that we use today is recombinant SPA (rSPA), which is produced in Escherichia coli. As rSPA is obtained by expressing SPA as an intracellular protein, its purification is tedious and time consuming. In order to obtain a large amount of highly purified rSPA with relative ease, we expressed SPA as a secretory form in the yeast Pichia pastoris. To increase the expression level of SPA and repress its proteolysis during fermentation, the cell density (OD600), temperature and pH at which SPA expression was induced as well as the induction time were optimized. The final yield of SPA obtained was about 8.8 g per liter of culture, which under the optimized fermentation condition, accounted for 80% of the total protein in the culture supernatant. The expressed SPA was purified from the culture supernatant by DEAE ion-exchange chromatography (IEC) after the supernatant was subjected to a desalting step. The purified SPA was resolved as a single band by SDS–PAGE and as a single peak by HPLC. Its identity was confirmed by MALDI-TOF MS and western-blot. Moreover, the protein also exhibited excellent affinity for IgG when tested with human IgG. The production and purification of SPA described in this study offers a new method for obtaining high level of SPA in relatively pure form that is suitable for practical application.

•Two hetero-assemblies, βCD1–Phe1, and βCD2–Phe1 were observed in water solution.•Distinct membrane-binding patterns for βCD, Phe, and their complexes were found.•Minor Phe trans-membrane energy barrier confirmed its membrane penetration ability.•Huge energy barriers for βCD-involved assemblies denied their membrane penetration.•Phe separation from βCD1–Phe1 was easier than that from βCD2–Phe1.The explicit-solvent molecular dynamic (MD) simulation and adaptive biased forces (ABF) methods were employed to systemically study the structural and thermodynamic nature of the β-cyclodextrin (βCD) monomer, phenanthrene (Phe) monomer, and their inclusion complexes in both the aqueous and membrane environments, aiming at clarifying the atomic-level mechanisms underlying in the CD-enhanced degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria. Simulations showed that βCD and Phe monomers could associate together to construct two distinctive assemblies, i.e, βCD1–Phe1 and βCD2–Phe1, respectively. The membrane-involved equilibrium simulations and the data of potential of mean forces (PMFs) further confirmed that Phe monomer was capable of penetrating through the membranes without confronting any large energy barrier, whereas, the single βCD and βCD-involved assemblies were unable to pass across the membranes. These observations clearly suggested that βCD only served as the carrier to enhance the bioavailability of Phe rather than the co-substrate in the Phe biodegradation process. The Phe-separation PMF profiles indicated that the maximum of the Phe uptake by bacteria would be achieved by the “optimal” βCD:Phe molar ratio, which facilitated the maximal formation of βCD1–Phe1 inclusion and the minimal construction of βCD2–Phe1 complex.Download full-size image

Enterobacter sp. LY402 is a bacterium isolated from polluted soil. It can efficiently degrade polychlorinated biphenyls (PCBs) under aerobic conditions. However, the degradation was limited when it comes to high chlorine or double para-substituted PCBs. Biphenyl dioxygenase (BDO) is the key enzyme in the PCBs biodegradation process. It has been confirmed that the α-subunit of the iron–sulfur protein of biphenyl 2,3-dioxygenase (BphA1) directly influenced catalytic activities and substrate specificity. To know the degradation characteristics of BDO to PCBs, we analyzed PCBs degradation abilities of BphA1 from Enterobacter sp. LY402 by experiment and molecular simulation. Firstly, the degradation experiment of PCBs was performed, and the degradation rate constants (k) were calculated. Then the three-dimensional model of LY402-BphA1 was constructed. Through further docking studies with 209 PCB congeners, the PCBs binding abilities of LY402-BphA1 were measured and some crucial active site residues were identified. Moreover, the molecular descriptors of PCBs were calculated and analyzed to determine the correlation of molecular properties and degradation. The results showed that the affinity energy of PCBs was well matched with the k values of the different number of chlorine substituents. The binding ability of BphA1 greatly affected the PCBs degradation abilities of BDO. Hydrophobic contact was the main interaction between the residues of active site and PCB substrates. The number and subposition of chlorine substituents would influence the PCBs binding ability of BphA1 significantly. Ser283, Val287, Gly321 and Tyr384 residues in the active site of LY402-BphA1 showed high variability, and the space limitation of the active site of BphA1 had negative influence on the PCBs binding affinity of BDO. The changes of physical, electronic and geometrical properties could influence degradation and binding affinity of PCBs. Analysis of structural information, binding affinity and influences of molecular properties could be used to direct further modification of BDO to enhance biodegradation of PCBs and other toxic compounds.

DDT (1,1,1-trichloro-2.2-bis(p-chlorophenyl)ethane), one of the most abused insecticides, is a highly hazardous component for both human health and environmental applications. The biodegradation of DDT into non-toxic, environmentally benign components is strongly limited by the poor bioavailability of DDT. In this work, we combined experiments and molecular simulations to examine the effect of three cyclodextrins (α-, β-, and γ-CD) on their structure-specific interactions with DDT, specifically in relation to DDT solubility and biodegradability. It was found that all three CDs were able to bind to DDT with their inner hydrophobic cavity and different binding affinities and orientations, demonstrating their ability to improve DDT solubility. Different from the strong binding between DDT and β-/γ-CDs via a fully DDT bury mode, α-CD had a relatively weak binding with DDT via a partial DDT bury mode, which allowed DDT to be readily disassociated from α-CD at the lipid membrane interface, followed by DDT permeation into and across the cell membrane. The different binding modes between DDT and CDs explain why only α-CD can promote the bioavailability and biodegradation of DDT by simultaneously increasing its aqueous solubility and membrane interaction. This work provides structural-based binding information for the further modification and optimization of these three CDs to enhance their solubility and biodegradability of DDT.

As a pathogenic toxin, bilirubin is generally removed from blood by hemoperfusion for the remission of liver disease or to gain time for patients waiting for liver transplantation. However, the development of bilirubin adsorbents with excellent mechanical properties, adsorption performance and hemocompatibility is still a considerable challenge. In this work, a heparin-modified chitosan/graphene oxide hybrid hydrogel (hep-CS/GH) has been developed for bilirubin adsorption using a lyophilization–neutralization–modification strategy. The as-prepared hybrid hydrogel displayed a unique foam-like porous structure and excellent mechanical flexibility. It was revealed that the incorporation of graphene oxide into the chitosan matrix enhanced both the compressive strength and the Young's modulus of the hybrid hydrogel, as well as its adsorption capacity for bilirubin. The maximum adsorption capacity of hep-CS/GH for bilirubin was 92.59 mg g−1, according to the Langmuir isotherm model. It was demonstrated that hep-CS/GH successfully competed with albumin, and could effectively adsorb bilirubin from a bilirubin-enriched serum. After the hydrogel was modified with heparin, protein adsorption, platelet adhesion and hemolysis were reduced, and the plasma clotting time was prolonged from 4.1 to 23.6 min, indicating the superior hemocompatibility of hep-CS/GH. Therefore, this study may pave the way for improving the performance of the adsorbent in removing blood toxins.

A metal–organic cerium tetrahedron having size constraints and cooperated interactions within its cavity was used to selectively recognize tryptophan over other natural amino acids and Trp-containing peptides. It was applied in quantificational detection of free tryptophan in serum.