Elution Of Proteins Research Articles

Evaluation of protein separations based on hydrophobic interaction chromatography using polyethylene terephthalate capillary-channeled polymer (C-CP) fiber phases

Poly(ethylene terephthalate) (polyester, PET) capillary-channeled polymer fibers are used as the stationary phase for the separation of a synthetic protein mixtures composed of cytochrome c, myoglobin, ribonuclease A, lysozyme, α-chymotrypsinogen A, α-chymotrypsin and holo-transferrin. Polyester is a useful phase for HIC, having a mixed alkyl and aromatic backbone to yield mildly hydrophobic properties, an aromatic functionality allowing for π-π interactions, and carboxylate end groups adding hydrophilicity. In addition, the lack of extraneous hydrophobic ligands or charged surface species alleviates secondary interactions between proteins and common support phases. Breakthrough experiments with lysozyme solution were operated at various loading flow rates (0.1–0.5 mL min−1) and protein concentrations (0.025–1.0 mg mL−1). Dynamic binding capacity (DBC) was plotted and results fit to two isotherm models: Langmuir and Freundlich. Column stability and reproducibility were tested via 10 continuous lysozyme load/elution cycles. Organic solvents, acetonitrile and isopropanol, were tested with proportions ranging from 0 to 40% in the elution buffer, aiding protein elution, with the best separation efficiency employing ∼15–20% organic modifier. Resolution was evaluated at solvent pH values between 5 and 7.5, flow rates ranging from 0.2 to 0.6 mL min−18−1) and at gradient slopes between 5 and 16.7% B min−1. Optimum resolution and fast analysis times were achieved employing a steep gradient using high linear velocities. Finally, a comparison of separation efficiency between a commercial HIC phase (TSKgel Phenyl-5 P W) and the PET fiber column is presented for a standard, six protein mixture, using optimal operation conditions for each column type.

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100μg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

Retention Behavior of Polyethylene Glycol and Its Influence on Protein Elution on Hydrophobic Interaction Chromatography Media

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface. The effect enhanced with increasing molecular weight of the polymer and hydrophobicity of the HIC media. Addition of PEG to the mobile phase reduced binding of proteins to HIC resins, which was demonstrated with two model systems: lysozyme (LYZ) and immunoglobulin G (IgG), and their mixtures. In case of LYZ, the presence of PEG caused reduction in the protein retention, whereas for IgG—a decrease in efficiency of the protein capture. The effect depended on the adsorption pattern of PEG; it was pronounced in the systems in which conformational changes of the polymer were suggested to occur.

Antibody immunoglobulin G1 and immunoglobulin G2a responses against some cystic fluid proteins of Cysticercus bovis in Balb/c mice.

Background and Aim:Immunoglobulin (Ig) G1 and IgG2a are the surrogate markers respectively for humoral and cellular immune responses of hosts against antigens including cystic fluid proteins of Cysticercus bovis. A study was conducted to investigate the IgG1 and IgG2a responses of Balb/c mice against some individual cystic fluid proteins of C. bovis in an effort to determine the roles of each protein in inducing the humoral and cellular immune responses in host.Materials and Methods:Individual p71, p31, and p14 proteins of C. bovis were purified by separation of the proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and elution of individual proteins from the gel. Six female Balb/c mice were immunized 4 times at 10-day intervals with the crude cystic fluid proteins, and sera were collected for the measurement of IgG1 and IgG2a levels against the individual proteins. Sera samples collected before the first immunization were used as negative antibody control, sera samples collected after the fourth immunization were used as positive antibody control, and crude cystic fluid protein was used as positive antigen control.Results:All immunized mice were immune to p71, p31, p14, and crude cystic fluid proteins of C. bovis. The crude cystic fluid proteins of C. bovis induced a higher IgG2a than IgG1 level following the first and the second immunizations but switched into a higher IgG1 than IgG2a level following the fourth immunization. Protein 71 kDa (p71) induced a higher IgG2a than IgG1 level following the fourth immunization. In contrast, p14 induced a higher IgG1 than IgG2a level following the fourth immunization. Low and balance IgG1 and IgG2a levels against p31 were observed following the first to the fourth immunizations.Conclusion:Using IgG1 and IgG2a levels as the surrogate markers, it appears that cystic fluid antigens of C. bovis induce both humoral and cellular immune responses in Balb/c mice. The p71 appears to be a better inducer of cellular immune response, whereas p14 is a better inducer of humoral immune response of mice.

Top-down protein identification using a time-of-flight mass spectrometer and data independent acquisition

Top-down mass spectrometry and direct dissociation of gas phase intact proteins have been demonstrated to be a powerful platform for identifying proteins from complex mixtures and for elucidating post-translational modifications (PTMs). Fragmentation of proteins in the atmospheric pressure/vacuum interface of the electrospray ionization mass spectrometer is an effective dissociation technique that can be utilized for on-line HPLC top-down analysis. We demonstrate the capability to perform intact protein identifications in a single-stage time-of-flight (TOF) mass spectrometer in a data independent (DIA) acquisition fashion by rapidly switching the in-source dissociation (ISD) energy during protein elution from a liquid chromatography (LC) column. The intact protein and product ion masses obtained at low and high ISD energies, respectively, were measured using a TOF mass analyzer. By coupling on-line protein separations to dissociation in the atmospheric pressure/vacuum interface region of the mass spectrometer, we identified proteins in simple complexity mixtures, including subunits from the human 20S proteasome complex, and PTMs such as phosphorylation and N-terminal acetylation events. This proof-of-principle study demonstrates that a data-independent pseudo-MS/MS method could be a relatively inexpensive platform for top-down MS.

Systematic Glycolytic Enzyme Activity Analysis from Human Serum with PEP Technology.

A functional proteomics technology was used to systematically monitor metabolic enzyme activities from resolved serum proteins produced by a modified 2-D gel separation and subsequent Protein Elution Plate, a method collectively called PEP. Both qualitative and quantitative differences in the metabolic enzyme activity could be detected between cancer patient and control group, providing excellent biomarker candidates for cancer diagnosis and drug development. This technology has a wide range of applications; it can be used for rapid functional protein purification and characterization as well as drug target identification and validation. The ability for the PEP technology to efficiently separate and recover functional proteins makes it useful for the analysis of any proteins and its variants; this is especially advantageous for enzyme families with large number of enzymes such as protein kinases, phosphatases, proteases, and metabolic enzymes.

Preparation of copolymer-grafted mixed-mode resins for immunoglobulin G adsorption

The mixed-mode resins for protein adsorption have been prepared by a novel strategy, copolymer grafting. Specially, the copolymer-grafted resins CG-M-A with two functional groups, 5-amino-benzimidazole (ABI) and methacryloxyethyltrimethyl ammonium chloride (METAC), have been prepared through surface-initiated activator generated by electron transfer for atom transfer radical polymerization of METAC and glycidyl methacrylate (GMA), followed by a ring-open reaction to introduce ABI. The charge and hydrophobicity of CG-MA resins could be controlled by manipulating the addition of METAC and GMA/ABI. Besides, METAC and ABI provided positive effects together in both protein adsorption and elution: dynamic binding capacity of human Immunoglobulin G (hIgG) onto CG-M-A resin with the highest ligand ratio of METAC to ABI is 46.8 mg∙g–1 at pH 9 and the elution recovery of hIgG is 97.0% at pH 5. The separation experiment showed that purity and recovery of monoclonal antibody from cell culture supernatant are 96.0% and 86.5%, respectively, indicating that copolymer-grafted mixed-mode resins could be used for antibody purification.

Selective release of overexpressed recombinant proteins from E. coli cells facilitates one-step chromatographic purification of peptide-tagged green fluorescent protein variants

The need for fast and convenient protein purification is enormous, not only on the industrial scale for the production of therapeutics but also in life science research. Most protein purification strategies require multiple steps, which are time consuming, cost and resource intensive. Selective release of the intracellular target product can considerably facilitate downstream processing by minimizing the HCP concentration before subsequent purification processes. Here, we present a simple method to selectively release soluble overexpressed proteins from E. coli with minimal laboratory equipment requirements. This method is based on the creation of pores in the cell envelope by a single freeze-thaw cycle. Proteins of various sizes were released by several 50 mM Tris pH 9.5 buffer washes with high yields. Consequently, the need for detergents and other additives were circumvented. Green fluorescent protein (GFP), was tagged with peptides of various charges and polarity. Releasing these GFP variants with our method enabled the successful one-step chromatographic purification on a quaternary amine resin despite the different isoelectric points of the target proteins. Up to 90% of the target protein elution peak during anion exchange chromatography was of >95% purity. This optimized process is more productive compared to multi-step purification procedures currently available in literature.

Use of mobile phase additives for the elution of bispecific and monoclonal antibodies from phenyl based hydrophobic interaction chromatography resins

Hydrophobic interaction chromatography (HIC) is routinely used in the purification of biopharmaceuticals such as antibodies. However, hydrophobic proteins can be difficult to elute resulting in low recovery of product thereby complicating early phase process development and potentially excluding the use of HIC resins for their manufacture. Mobile phase additives such as hexylene glycol and arginine facilitate protein elution from resins including HIC; therefore, these additives were evaluated toward the recovery and purification of bispecific and monoclonal antibodies from Phenyl Sepharose HP and Capto Phenyl ImpRes resins. The influences of gradient versus step elution as well as superficial linear velocity on product quality were evaluated. Improved protein recovery and reduction of both soluble product aggregate and host cell protein were observed for the tested antibodies with both hexylene glycol and arginine. Furthermore, the impact of salt removal from the HIC load on protein-resin binding was examined providing opportunities to minimize processing time. This method was successfully scaled using a Phenyl Sepharose HP (5 cm i.d. × 20.0 cm) and Capto Phenyl ImpRes (3.2 cm i.d. × 21.4 cm) column demonstrating potential for manufacturing purposes.

Investigating the impact of aromatic ring substitutions on selectivity for a multimodal anion exchange prototype library

The increasing prevalence of low pI non-mAb therapeutics as well as current challenges in mAb-aggregate separations and low recoveries motivate further development in the multimodal anion exchange (MM AEX) space. In this work, linear salt gradient experiments at pH 7 were used to evaluate the retention of model proteins (with pI from 3.4 to 6.8) in 17 novel MM AEX prototype systems. The ligands were organized into three series. Series 1 extended previous work in multimodal ligand design and included a hydroxyl variant and linker length variants. Series 2 and 3 investigated the nature of hydrophobicity in MM AEX systems by adding hydrophobic (series 2) or fluorine (series 3) substituents to a solvent exposed phenyl ring. Compared to the commercial resin Capto Adhere, the series 1 and 3 ligands exhibited weaker binding, while some of the series 2 aliphatic prototypes showed dramatically increased retention and unique selectivities. Within series 1, the model proteins eluted earlier in the gradient as the charge-hydrophobic group distance on the ligand was increased from 4.9 Å to 8.5 Å. For the aliphatic variants in series 2, proteins that eluted early in the salt gradient were not affected by the increase in ligand hydrophobicity, while the later eluting proteins bound stronger as the length of the aliphatic substituent increased. The series 3 variants indicated that phenyl ring fluorination created subtle changes in protein elution in these MM AEX systems. Retention data from the three series was used to generate a partial least squares QSAR model based on both protein and ligand descriptors which accurately predicted protein retention with a training R2 of 0.81 and a test R2 of 0.76. The retention characteristics of some prototypes such as the earlier elution and unique selectivities compared to Capto Adhere suggest that they could potentially provide unique selectivities and increased recovery for the downstream processing of both mAb and non-mAb biotherapeutics.

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Effects of hydrogenated TiO2 nanotube arrays on protein adsorption and compatibility with osteoblast-like cells.

BackgroundModified titanium (Ti) substrates with titanium dioxide (TiO2) nanotubes have broad usage as implant surface treatments and as drug delivery systems.MethodsTo improve drug-loading capacity and accelerate bone integration with titanium, in this study, we hydrogenated anodized titanium dioxide nanotubes (TNTs) by a thermal treatment. Three groups were examined, namely: hydrogenated TNTs (H2-TNTs, test), unmodified TNTs (air-TNTs, control), and Ti substrates (Ti, control).ResultsOur results showed that oxygen vacancies were present in all the nanotubes. The quantity of -OH groups greatly increased after hydrogenation. Furthermore, the protein adsorption and loading capacity of the H2-TNTs were considerably enhanced as compared with the properties of the air-TNTs (P<0.05). Additionally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to investigate the interactions of TNTs with proteins. During the protein-loading process, the H2-TNTs not only enabled rapid protein adsorption, but also decreased the rate of protein elution compared with that of the air-TNTs. We found that the H2-TNTs exhibited better biocompatibility than the air-TNT and Ti groups. Both cell adhesion activity and alkaline phosphatase activity were significantly improved toward MG-63 human osteoblast-like cells as compared with the control groups (P<0.05).ConclusionWe conclude that hydrogenated TNTs could greatly improve the loading capacity of bioactive molecules and MG-63 cell proliferation.

Comparison of Techniques for Preimplantation Treatment of Osteochondral Allograft Bone.

Articular defects are a major problem with few effective treatment options. Osteochondral allograft (OCA) transplantation can be an effective treatment; however, lack of OCA bone integration can cause failure. This controlled laboratory study was designed to compare clinically applicable methods for marrow element removal and enhanced delivery of bone marrow aspirate concentrate (BMC) to OCA bone. We hypothesized that compressed carbon dioxide (CO2) treatment of OCA bone would result in significantly better marrow element removal, significantly more retention and distribution of viable osteoprogenitor cells, and significantly higher osteoinductive protein elution from OCAs compared with other preimplantation treatments. Fresh humeral heads (n = 24) were harvested and stored for 14 days, then randomly assigned to treatment based on marrow element removal and bone treatment: (standard of care [SOC]) (n = 4) - SOC high-pulse saline lavage, no BMC; (BMC) (n = 5) - saline lavage then canine BMC; (Drill + BMC) (n = 5) - 1.1 mm drill-hole immediately subchondral then saline lavage then BMC injection through drill hole; (Carb + BMC) (n = 5) - saline lavage then CO2 then BMC; or (Saline-Carb + BMC) (n = 5) - saline lavage and CO2 together then BMC. Treated OCAs were cultured for 14 days. On day 3, media were collected, centrifuged to isolate cells, and replaced. Cells were cultured for 11 days for colony forming unit (CFU) determination. OCA media were collected on days 7 and 14 of culture for analysis. On day 14, each graft was assessed for viable cell retention and distribution, and bone marrow element removal. BMC had significantly higher (p = 0.001) viable cell distribution compared with the SOC, Drill + BMC, Carb + BMC, and Saline-Carb + BMC groups. BMC and Drill + BMC had significantly higher (p < 0.05) CFUs than SOC, Carb + BMC, and Saline-Carb + BMC. Drill + BMC and Carb + BMC had the highest media concentrations of the osteoinductive biomarkers. The Carb + BMC and Saline-Carb + BMC groups were associated with significantly superior marrow element removal (p < 0.02) compared with the SOC, Drill + BMC, and BMC groups. Saline irrigation plus saturation with autogenous BMC appears to be the most advantageous preimplantation treatment for OCA transplantation.

Modeling and simulation of protein elution in linear pH and salt gradients on weak, strong and mixed cation exchange resins applying an extended Donnan ion exchange model

Process development and characterization based on mathematic modeling provides several advantages and has been applied more frequently over the last few years. In this work, a Donnan equilibrium ion exchange (DIX) model is applied for modelling and simulation of ion exchange chromatography of a monoclonal antibody in linear chromatography. Four different cation exchange resin prototypes consisting of weak, strong and mixed ligands are characterized using pH and salt gradient elution experiments applying the extended DIX model. The modelling results are compared with the results using a classic stoichiometric displacement model. The Donnan equilibrium model is able to describe all four prototype resins while the stoichiometric displacement model fails for the weak and mixed weak/strong ligands. Finally, in silico chromatogram simulations of pH and pH/salt dual gradients are performed to verify the results and to show the consistency of the developed model.

Self-Reporting Photoluminescent Porous Silicon Microparticles for Drug Delivery.

A porous Si (pSi) microparticle-based delivery system is investigated, and the intrinsic luminescence from the particles is employed as a probe to monitor the release of a model protein payload, bovine serum albumin (BSA). The microparticles consist of a core Si skeleton surrounded by a SiO2 shell. Two types of pSi are tested, one with smaller (10 nm) pores and the other with larger (20 nm) pores. The larger pore material yields a higher mass loading of BSA (3 vs 20%). Two different methods are used to load BSA into these nanostructures: the first involves loading by electrostatic physisorption, and the second involves trapping of BSA in the pSi matrix by local precipitation of magnesium silicate. Protein release from the former system is characterized by a burst release, whereas in the latter system, release is controlled by dissolution of the pSi/magnesium silicate matrix. The protein release characteristics are studied under accelerated (0.1 M aqueous KOH, 21 °C) and physiologically relevant (phosphate-buffered saline, pH 7.4, 37 °C) conditions, and the near-infrared photoluminescence signal from the pSi skeleton is monitored as a function of time and correlated with protein release and silicon dissolution. The thickness of the Si core and the SiO2 shell are systematically varied, and it is found that the luminescence signature can be tuned to provide a signal that either scales with protein elution or that changes rapidly near the end of useful life of the delivery system. Although payload release and particle dissolution are not driven by the same mechanism, the correlations between luminescence and payload elution for the various formulations can be used to define design rules for this self-reporting delivery system.

Protein adsorption onto monoliths: A surface energetics study.

This part of work was done to explore the basic understanding of the adsorption chromatography by determining the interaction of selected model proteins (n = 5) to monolithic chromatographic materials, with varying densities of butyl and phenyl ligands. Surface energetics approach was applied to study the interaction behavior. The physicochemical properties of the proteins and monolithic chromatographic materials were explored by contact angle and zeta potential values. These values were used to study protein to monolith interaction under various operating conditions. Surface energetics approach allowed the calculation of interaction energy as a function of distance, i.e. energy minimum values. Calculations were performed at various conditions to analyze the effect of major operating parameters on the interaction strength. The interaction strength exposed the hydrophobic nature of the monoliths which increases with increasing ligand density. Further, interaction energy of proteins were higher with monolith with butyl ligand compared to monolith with phenyl ligand. For instance, lactoferrin interaction to monoliths with butyl represents more interaction, i.e. 24.38 kT as compared to monoliths with phenyl i.e. 23.28 kT, keeping lambda as 0.2 nm and salt concentration as 100mM of ammonium sulphate. Hence, more energy and time will be consumed for elution of proteins immobilized to monoliths with butyl. Similarly, the effect of solid surface for proteins immobilization, effect of ligand density and effect of lambda showed some interesting insights on the interaction behavior. The knowledge generated from the present work will help in the basic understanding as well as development of an efficient, low cost downstream processing design and may mimic the real chromatographic experiments.

A rapid and accurate approach for prediction of interactomes from co-elution data (PrInCE)

BackgroundAn organism’s protein interactome, or complete network of protein-protein interactions, defines the protein complexes that drive cellular processes. Techniques for studying protein complexes have traditionally applied targeted strategies such as yeast two-hybrid or affinity purification-mass spectrometry to assess protein interactions. However, given the vast number of protein complexes, more scalable methods are necessary to accelerate interaction discovery and to construct whole interactomes. We recently developed a complementary technique based on the use of protein correlation profiling (PCP) and stable isotope labeling in amino acids in cell culture (SILAC) to assess chromatographic co-elution as evidence of interacting proteins. Importantly, PCP-SILAC is also capable of measuring protein interactions simultaneously under multiple biological conditions, allowing the detection of treatment-specific changes to an interactome. Given the uniqueness and high dimensionality of co-elution data, new tools are needed to compare protein elution profiles, control false discovery rates, and construct an accurate interactome.ResultsHere we describe a freely available bioinformatics pipeline, PrInCE, for the analysis of co-elution data. PrInCE is a modular, open-source library that is computationally inexpensive, able to use label and label-free data, and capable of detecting tens of thousands of protein-protein interactions. Using a machine learning approach, PrInCE offers greatly reduced run time, more predicted interactions at the same stringency, prediction of protein complexes, and greater ease of use over previous bioinformatics tools for co-elution data. PrInCE is implemented in Matlab (version R2017a). Source code and standalone executable programs for Windows and Mac OSX are available at https://github.com/fosterlab/PrInCE, where usage instructions can be found. An example dataset and output are also provided for testing purposes.ConclusionsPrInCE is the first fast and easy-to-use data analysis pipeline that predicts interactomes and protein complexes from co-elution data. PrInCE allows researchers without bioinformatics expertise to analyze high-throughput co-elution datasets.

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Facile and green fabrication of cation exchange membrane adsorber with unprecedented adsorption capacity for protein purification

Fabricating membrane adsorbers with high adsorption capacity and appreciable throughput for the separation and purification of protein products is challenging in biomedical and pharmaceutical industries. Herein, we report the synthesis of a novel membrane adsorber by functionalizing a nylon microfiltration membrane with alginate dialdehyde (ADA) followed by sulphonic addition, without any solvent usage, and its successful application in the purification of lysozyme. Taking advantage of abundant dual cation exchange (CEX) groups on sulphonic-ADA (S-ADA) ligands, this novel S-ADA-nylon membrane adsorber showed an unprecedented static binding capicity of 286mg/mL for lysozyme adsorption. Meanwhile, the prepared membrane adsorber could be easily regenerated (complete protein elution) under mild conditions and be reused at least for five times. Featured with a unique selectivity, the S-ADA-nylon membrane also captured lysozyme from chicken egg white solution with a high purity (100%) and a high recovery of 98%. The purified lysozyme showed similar specific activity as commercial product. The present work provides a facile, green and low-cost approach for the preparation of high-performance membrane adsorbers, which has a great potential in protein production.

Optimized protocol for whole organ decellularization

BackgroundThe idea of tissue decellularization to gain matrices for tissue engineering is promising. The aim of the present study is to establish a safe and reproducible protocol for solid tissue decellularization that prevents the architecture of the matrix with the inherent vascular network.MethodsThe study was performed in rat kidneys which were decellularized by a SDS-based perfusion protocol. Perfusion time and SDS concentration were systematically changed to obtain the shortest and most gentle protocol that leads to complete decellularization.ResultsWe investigated kinetics of protein elution, decellularization success, and remaining cell toxicity. This resulted in a reproducible protocol, leading to safe decellularization with prevention of the inherent vascular network, without remaining detectable cell toxicity. The established protocol leads to solid tissue decellularization in only 7 h, which is by far shorter than the previously published methods.ConclusionThe established technique has the potential to become a relevant platform technology for tissue engineering of solid tissues. It provides a solution for the yet-unsolved problem of vascularization.