High-throughput Protein Separation Research Articles

Investigation of differences in the protein transport capability of homochiral nanochannels

Chiral recognition plays a crucial role in the normal functioning of biological systems. The recognition of proteins in chiral environments underpins many fundamental life processes. Taking advantage of the distinct interactions between different proteins and chiral environments, this study presents the design of homochiral metal–organic framework (MOF)/nanochannels based membrane separator, enabling highly selective and high-throughput protein separation. The chiral separation membrane was fabricated by employing TiO2 nanochannel membrane (TM) as the supporting membrane and Ti ion source. Using terephthalic acid (BDC) and d/l-phenylalanine (DP/LP) as ligands, a layered TiMOF (MIL-125(Ti) used in this study) incorporating chiral selector molecules (named as DP/M and LP/M) were synthesized in situ within the TiO2 nanochannels. The bovine serum albumin (BSA) adsorption capacity of DP/M decorated TM was demonstrated to be 2.8 times higher than that of LP/M decorated TM, and was found to be related to the content of the chiral selector DP in the separation membrane. Furthermore, different recognition abilities by the chiral channels were observed for proteins with different isoelectric points. Based on a comprehensive exploration of the variations in the interaction forces between protein and chiral selector molecules, a nanoscale chromatography column-like separation model was proposed. The chiral separation membranes designed in this study provide a new platform for understanding the interactions between chiral compounds and proteins, and open up new avenues for fabricating chiral bio-interface materials, elucidating the role of chiral recognition in biological systems, and developing novel biomaterials and devices.

Eco-friendly and underwater superelastic cellulose nanofibrous aerogels for efficient capture and high-throughput protein separation

High-performance and environmentally compatible chromatographic media with superb adsorption capacity and large processing throughput is key to efficient protein separation. Herein, composite aerogels (CCS-NFAs) of cellulose nanofibrous and chitosan (CCS) with 3D lamellar structure were developed by freeze-drying method for adsorptive separation of protein. The CCS-NFAs exhibited good under water strength (25.2 kPa at 60 % compression strain) and underwater superelasticity despite being highly porous (over 98%) and lightweight (10.94 mg cm−3). The maximum adsorption capacity for bovine serum albumin (BSA) was calculated to be about 1269 mg g−1, and the corresponding dynamic adsorption capability was as high as 914 mg g−1 with a filling mass of 20 mg at a flow rate of 30 mL h−1, compared favorably against the state-of-the-art ion-exchange chromatography materials. The superb adsorptive capacity was due to the involvement of chemical adsorption as confirmed by the pseudo-second-order kinetic model and Langmuir model fitting of the experimental data. Additionally, the highly oriented large open-cells in the material enabled ultrahigh water flux at about 2.2 × 104 L m-2h−1, which showed no significant attenuation in flux or removal efficiency. The successful preparation of CCS-NFAs contributes to protein purification engineering by offering an eco-friendly high-performance chromatographic media.

High-surface-area interconnected macroporous nanofibrous cellulose microspheres: a versatile platform for large capacity and high-throughput protein separation

With the rapid development of biopharmaceutical industry, conventional chromatographic media cannot meet the increasing demand for high-quality proteins. Here, high-surface-area interconnected macroporous nanofibrous cellulose microspheres (NCMs) are fabricated by combining emulsion technique and template method. The macroporous nanofibrous structure of NCMs contributes to the fast transfer of liquid and improves processing efficiency, and the high specific surface area endows NCMs with numerous active sites for the immobilization of adsorption ligands. Benefiting from the advantages of microstructure, NCMs are highly suitable as a versatile adsorbent platform for highly effective protein separation. As a proof of concept, we prepare three NCM-based protein adsorbents with different adsorption mechanisms including anion ion-exchange adsorbent, Cu2+-chelating affinity adsorbent, and hydrophobic interaction adsorbent, which all exhibit high adsorption capacity and fast adsorption kinetic, outstanding recyclability and structure stability for targeted protein, much superior to commercial and most of previously reported same type of adsorbents. We expect that the NCM would provide a promising candidate for the design and construction of high-performance protein adsorbent.

Recent advances in protein chromatography with polymer-grafted media

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.

A Multichannel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High-Throughput Protein Separation and Characterization.

We developed a multichannel gel electrophoresis system that continuously collects fractions as protein bands migrate to the bottom of gel columns. The device uses several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "counter-free-flow" elution technique allows continuous and simultaneous fraction collection from multiple channels at low cost. Using the system with SDS-PAGE, 300μg samples of protein can be separated and eluted into 48-96 fractions over a mass range of 10-150kDa in 2.5h. Each eluted protein can be recovered at 50% efficiency or higher in ~500μL. The system can also be used for native gel electrophoresis, but protein aggregation limits the loading capacity to about 50μg per channel and reduces resolution. This system has the potential to be coupled with mass spectrometry to achieve high-throughput protein identification.

Nanobiotechnology: (Small 34/2016)

SmallVolume 12, Issue 34 p. 4774-4774 Back CoverFree Access Nanobiotechnology: (Small 34/2016) First published: 07 September 2016 https://doi.org/10.1002/smll.201670173AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract The State Key Laboratory of Biochemical Engineering (SKLBE) in the Institute of Processing Engineering, CAS is highlighted on the back cover. The laboratory has established micro-pore emulsification, multi-shell formation and self-assembly technologies to bring great advances in nanobiotechnology. The developed nanoparticles or devices, with uniform-sizes and controlled structures, have exerted themselves in the fundamental research as well as the industrial applications on enzyme immobilization, high-throughput protein separation medium, controlled release and targeted diagnoses, and delivery of antigens, siRNA, and anti-cancer drugs. Volume12, Issue34Special Issue: Advances in NanobiotechnologySeptember 14, 2016Pages 4774-4774 RelatedInformation

Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins

Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been previously studied as stationary phases for reversed phase and affinity protein separations. In this study, surface modified PET C-CP fibers were evaluated for the anion exchange separation of proteins. The native PET C-CP fibers were aminated using polyethylenimine (PEI) followed by a 1,4-butanediol diglycidyl ether (BUDGE) cross-linking step. Subsequent PEI/BUDGE treatments can be employed to further develop the polyamine layer on the fiber surfaces. The PEI densities of the modified fibers were quantified through the ninhydrin reaction, yielding values of 0.43–0.89μmolg−1. The surface modification impact on column permeability was found to be 0.66×10−11 to 1.33×10−11m2, depending on the modification time and conditions. The dynamic binding capacities of the modified fiber media were determined to be 1.99–8.54mgmL−1 bed volume, at linear velocities of 88–438cmmin−1 using bovine serum albumin as the model protein. It was found that increasing the mobile phase linear velocity (up to 438cmmin−1) had no effect on the separation quality for a synthetic protein mixture, reflecting the lack of van Deemter C-term effects for the C-CP fiber phase. The low-cost, easy modification method and the capability of fast protein separation illustrate great potential in the use of PEI/BUDGE-modified PET C-CP fibers for high-throughput protein separation and downstream processing.

A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.

The Selective Protein Separations with Polyaminofunctionality on Controlled Silica Coating-Layers of Magnetic Nanoparticles

This work reported the development of the high throughput protein separation process with molecularly assembled silica-coated magnetic nanoparticles as a function of amino group numbers such as mono-, di-, and tri-aminofunctionality, in which the range of silica coating thicknesses were optimized to be interacted with protein. The protein separation efficiency was demonstrated as a function of each aminofunctional group and the particle sizes of the silica coated magnetic nanoparticles. The particles were prepared by the chemical precipitation of Fe2+ and Fe3+ salts with a molar ratio of 1:2 under basic solution. The silica coated magnetic nanoparticles were directly produced by the sol-gel reaction of a tetraethyl orthosilicate (TEOS) precursor, in which the coating layer serves as a biocompatible and versatile group for further biomolecular functionalization. To effectively capture the proteins, silica coated magnetic nanoparticles need to be functionalized reproducibly on the silica surface, and three kinds of amino functional groups were adapted as a function of number of amine using the mono-, di-, and tri-aminopropylalkoxysilanes.

Inner ear proteomics: A fad or hear to stay

Proteomics, the large-scale analysis of the structure and function of proteins, as well as of protein–protein interactions, has evolved into a major component of ‘systems analysis’. This requires the integration of information from different sources and at multiple levels, and involves two distinct parameters, (1) high-throughput protein separation, identification, and characterization, and (2) the extension of the obtained analytical data for the determination of the physiological function. The inner ear poses exceptional challenges to the study of proteomics because of its minute size, poor accessibility, association with complex fluid spaces, and diversity of cell types. Various approaches to the study of proteomics of the inner ear are presented, and success stories, noteworthy failures and what lies ahead, will be discussed.