Membrane separations in biotechnology

Membranes have always been an integral part of biotechnology processes. The sterile filtration of fermentation media, purification buffers, and protein product pools is standard practice in industry. Microfiltration is also used extensively for medium exchange and harvest. Ultrafiltration can be found in virtually every biotechnology process. A significant number of mammalian cell processes use filtration as an integral part of the overall strategy for viral clearance. Depth filters have also seen widespread use for the clarification of both mammalian and bacterial feed streams. Improvements in membrane technology are now focused on high-resolution applications, including improved protein-virus separation, protein purification by high-performance tangential flow filtration and enhanced membrane chromatography. These developments will allow membranes to play an important role in the evolution of the next generation of biotechnology processes.     

GM1 asymmetry in the membrane stabilizes pores

Cell membranes are highly asymmetric and their stability against poration is crucial for survival. We investigated the influence of membrane asymmetry on electroporation of giant unilamellar vesicles with membranes doped with GM1, a ganglioside asymmetrically enriched in the outer leaflet of neuronal cell membranes. Compared with symmetric membranes, the lifetimes of micronsized pores are about an order of magnitude longer suggesting that pores are stabilized by GM1. Internal membrane nanotubes caused by the GM1 asymmetry, obstruct and additionally slow down pore closure, effectively reducing pore edge tension and leading to leaky membranes. Our results point to the drastic effects this ganglioside can have on pore resealing in biotechnology applications based on poration as well as on membrane repair processes.     

Use of membrane operation for refining gibberellinic acid

Synthetic membranes are increasingly used in biotechnology. The possibilities of influencing the performance by membrane operation and changing the performance by physical modification were discussed. With cellulose-2,5-acetate membranes it was shown that the modification leads to an increase in the concentration of gibberellinic acid.     

Formation of a bilayer lipid membrane on rigid supports: an approach to BLM-based biosensors

Sensory transduction in living cells is thought to involve a change of electrical parameters at the receptor membrane following specific binding events at the membrane surface. Because of the complexity of the biomembrane structure and the environmental factors associated with it, experimental bilayer lipid membranes (BLMs) have been employed for elucidation of processes at the membrane level. This is because the BLM system can be easily probed by a host of powerful and sensitive electrochemical methods. Further, recent advances in microelectronics and biotechnology suggest that the development of a BLM-based electrochemical biosensor may be possible. This paper describes the use of bilayer lipid membranes on solid substrates for analysis of sensor development problems, with relevance to a possible novel type of biomolecular device. Some electrical parameters of the new structure were measured and compared to usual BLM results. The advantages of the self-assembled structure, together with the measuring system, are discussed in terms of stability and sensitivity.     

The role of ion-lipid interactions and lipid packing in transient defects caused by phenolic compounds

The transient disruption of membranes for the passive permeation of ions or small molecules is a complex process relevant to understanding physiological processes and biotechnology applications. Phenolic compounds are widely studied for their antioxidant and antimicrobial properties, and some of these activities are based on the interactions of the phenolic compound with membranes. Ions are ubiquitous in cells and are known to alter the structure of phospholipid bilayers. Yet, ion-lipid interactions are usually ignored when studying the membrane-altering properties of phenolic compounds. This study aims to assess the role of Ca²⁺ ions on the membrane-disrupting activity of two phenolic acids and to highlight the role of local changes in lipid packing in forming transient defects or pores. Results from tethered bilayer lipid membrane electrical impedance spectroscopy experiments showed that Ca²⁺ significantly reduces membrane disruption by caffeic acid methyl ester and caffeic acid. As phenolic acids are known metal chelators, we used UV-vis and fluorescence spectroscopy to exclude the possibility that Ca²⁺ interferes with membrane disruption by binding to the phenolic compound and subsequently preventing membrane binding. Molecular dynamics simulations showed that Ca²⁺ but not caffeic acid methyl ester or caffeic acid increases lipid packing in POPC bilayers. The combined data confirm that Ca²⁺ reduces the membrane-disrupting activity of the phenolic compounds, and that Ca²⁺-induced changes to lipid packing govern this effect. We discuss our data in the context of ion-induced pores and transient defects and how lipid packing affects membrane disruption by small molecules.     

Performance characteristics of nanoporous carbon membranes for protein ultrafiltration

Nanoporous carbon membranes could be very attractive for applications of ultrafiltration in the biotechnology industry because of their greater mechanical strength and longer membrane life. The objective of this study was to obtain quantitative data on the performance characteristics of nanoporous carbon membranes formed within a stainless steel support that was first modified by deposition of silica particles within the macroporous support. The nanoporous carbon membrane effectively removed small solutes from a protein solution using diafiltration, with performance comparable to that of commercial polymeric membranes. Protein fouling was evident, although the nanoporous carbon membranes were easily regenerated; cleaning with 0.5 N NaOH at 50 degrees C completely restored the water permeability for multiple cycles. The nanoporous carbon membranes were also compatible with steam sterilization. Significant increases in process flux could be obtained using periodic back-pulsing, with no evidence of any structural alterations in the membrane. These results clearly demonstrate the potential benefits and opportunities for using nanoporous carbon membranes for protein ultrafiltration.     

Effects of high pressure on lipids and biomembranes for understanding high-pressure-induced biological phenomena.

This review covers high pressure effects on lipids, lipid bilayers, and biochemical observations recently found in the field of high-pressure bioscience and biotechnology including deep-sea microbiology and food science. To explain these phenomena in a unified model, recent studies of physical and chemical properties of artificial membranes and natural membranes are summarized. On the basis of this newly described knowledge, high pressure effects on biochemical events are considered at the molecular level and concluded that high pressure induces decreases in biomembrane fluidity and phase transitions that result in breakage of the membrane, and finally, leads to the destruction of bilayer membrane accompanied by denaturation of membrane-associated proteins.     

Simulations of electrophoretic RNA transport through transmembrane carbon nanotubes

The study of interactions between carbon nanotubes and cellular components, such as membranes and biomolecules, is fundamental for the rational design of nanodevices interfacing with biological systems. In this work, we use molecular dynamics simulations to study the electrophoretic transport of RNA through carbon nanotubes embedded in membranes. Decorated and naked carbon nanotubes are inserted into a dodecane membrane and a dimyristoylphosphatidylcholine lipid bilayer, and the system is subjected to electrostatic potential differences. The transport properties of this artificial pore are determined by the structural modifications of the membrane in the vicinity of the nanotube openings and they are quantified by the nonuniform electrostatic potential maps at the entrance and inside the nanotube. The pore is used to transport electrophoretically a short RNA segment and we find that the speed of translocation exhibits an exponential dependence on the applied potential differences. The RNA is transported while undergoing a repeated stacking and unstacking process, affected by steric interactions with the membrane headgroups and by hydrophobic interaction with the walls of the nanotube. The RNA is structurally reorganized inside the nanotube, with its backbone solvated by water molecules near the axis of the tube and its bases aligned with the nanotube walls. Upon exiting the pore, the RNA interacts with the membrane headgroups and remains attached to the dodecane membrane while it is expelled into the solvent in the case of the lipid bilayer. The results of the simulations detail processes of molecular transport into cellular compartments through manufactured nanopores and they are discussed in the context of applications in biotechnology and nanomedicine.     

Flotillins functionally organize the bacterial membrane

Proteins and lipids are heterogeneously distributed in biological membranes. The correct function of membrane proteins depends on spatiotemporal organization into defined membrane areas, called lipid domains or rafts. Lipid microdomains are therefore thought to assist compartmentalization of membranes. However, how lipid and protein assemblies are organized and whether proteins are actively involved in these processes remains poorly understood. We now have identified flotillins to be responsible for lateral segregation of defined membrane domains in the model organism Bacillus subtilis. We show that flotillins form large, dynamic assemblies that are able to influence membrane fluidity and prevent condensation of Laurdan stained membrane regions. Absence of flotillins in vivo leads to coalescence of distinct domains of high membrane order and, hence, loss of flotillins in the bacterial plasma‐membrane reduces membrane heterogeneity. We show that flotillins interact with various proteins involved in protein secretion, cell wall metabolism, transport and membrane‐related signalling processes. Importantly, maintenance of membrane heterogeneity is critical for vital cellular processes such as protein secretion.     

Fabrication and electromechanical characterization of freestanding asymmetric membranes

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.     

Specialised plasma membranes in the preovulatory follicle op the fowl

Summary In the hen's follicle processes from the zona granulosa cells extend into the zona radiata. The terminal plasma membrane of these processes has a total thickness of around 160 Å and consists of five layers. Small granules spaced at regular intervals are attached to the cytoplasmic aspect of the inner layer by short stalks. In the 2 mm and 7 mm follicles the plasma membrane of the ovum facing the specialised terminal membranes has a striated appearance and shows a regular arrangement of granules attached by stalks to both its inner and outer aspects. The terminal and striated membranes are separated by an interval although there are areas of closer contact. In the 15 mm and 35 mm pre-ovulatory follicles the plasma membrane round the whole surface of the ovum is now typical striated membrane with bristles and attached granules. No explanation can be given at present of the function of the terminal membranes of the granulosa processes. They may indicate some change in the permeability permitting the intercellular diffusion of particles. It is suggested that the striated ovum plasma membrane is associated with the adsorption and transport of substances into the ovum for yolk synthesis.     

Intramembrane electrostatic interactions destabilize lipid vesicles

Membrane stability is of central concern in many biology and biotechnology processes. It has been suggested that intramembrane electrostatic interactions play a key role in membrane stability. However, due primarily to a lack of supporting experimental evidence, they are not commonly considered in mechanical analyses of lipid membranes. In this paper, we use the micropipette aspiration technique to characterize the elastic moduli and critical tensions of lipid vesicles with varying surface charge. Charge was induced by doping neutral phosphatidylcholine vesicles with anionic lipids phosphatidylglycerol and phosphatidic acid. Measurements were taken in potassium chloride (moderate ion-lipid binding) and tetramethylammonium chloride (low ion-lipid binding) solutions. We show that inclusion of anionic lipid does not appreciably alter the areal dilation elasticity of lipid vesicles. However, the tension required for vesicle rupture decreases with increasing anionic lipid fraction and is a function of electrolyte composition. Using vesicles with 30% charged (i.e., unbound) anionic lipid, we measured critical tension reductions of 75%, demonstrating the important role of electrostatic interactions in membrane stability.     

Membranes and bioreactors: a technical challenge in biotechnology

Integrating the properties of synthetic membranes with biological catalysts such as cells and enzymes forms the basis of an exciting new technology called membrane bioreactors. The impetus behind this marriage comes from the recent spectacular advances in recombinant DMA and cell fusion technologies and the need to develop competitive bioprocessing schemes to produce complex and active biological molecules. The advantages and limitations of using membrane bioreactors for entrapping whole cells and enzymes are reviewed. Various membrane configurations such as microcapsules, hollow fibers, and flat sheets are compared. Several different entrapped membrane bioreactors, including single, laminated and microporous, for the conversion of optically active enantiomers are described. As with new and exciting technologies, the future of membrane bioreactors in biotechnology will depend on their ability to produce desired molecules at competitive costs.     

Membrane technology for purification of therapeutic proteins

Membrane systems are used throughout the downstream purification in the commercial production of high value therapeutic proteins. Over the last two decades, new membranes, modules, and systems have been developed specifically to meet the requirements of the biotechnology industry. These developments have been facilitated by an improved fundamental understanding of: (1) the effects of electrostatic interactions and concentration polarization on protein transmission during ultrafiltration and (2) the role of membrane morphology on protein fouling during both sterile and virus filtration. This perspective highlights some of the key work in this area and provides insights into possible future improvements in membrane technology for the purification of recombinant therapeutic proteins. Biotechnol. Bioeng. 2009;103: 227–230. © 2009 Wiley Periodicals, Inc.     

Resolving the kinetics of lipid,protein and peptide diffusion in membranes

Abstract

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

Unique natural-protein hollow-nanofiber membranes produced by weaver ants for medical applications

We report the properties of unique natural-protein hollow-nanofiber membranes produced by weaver ants (Oecophylla smaragdina) and the potential of using the nanofiber membranes for medical applications. Although natural proteins such as silk and collagen have been used to produce electrospun nanofibers for medical applications, there are no reports on producing hollow nanofibers from proteins. Hollow nanofibers are expected to have unique properties such as high drug loading. Weaver ant larvae extrude proteins in the form of nanofibers that are hollow and the adult ants build the nests using the hollow nanofibers. It was found that the nanofiber membranes are composed of fibers with average diameters of 450 nm. The membranes have tensile strength of about 4 MPa, high elongation of about 31% and modulus of 31 MPa, better than any protein nanofiber membrane reported so far. The membranes withstand rigorous boiling in weak alkali, show good attachment and proliferation of osteoblasts and can load up to 4.7 times higher drugs compared to common silk. These features make ant nanofiber membranes unique and preferable for medical and biotechnology industries.     

Switch-like Responses of Two Cholesterol Sensors Do Not Require Protein Oligomerization in Membranes

Many cellular processes are sensitive to levels of cholesterol in specific membranes and show a strongly sigmoidal dependence on membrane composition. The sigmoidal responses of the cholesterol sensors involved in these processes could arise from several mechanisms, including positive cooperativity (protein effects) and limited cholesterol accessibility (membrane effects). Here, we describe a sigmoidal response that arises primarily from membrane effects due to sharp changes in the chemical activity of cholesterol. Our models for eukaryotic membrane-bound cholesterol sensors are soluble bacterial toxins that show an identical switch-like specificity for endoplasmic reticulum membrane cholesterol. We show that truncated versions of these toxins fail to form oligomers but still show sigmoidal binding to cholesterol-containing membranes. The nonlinear response emerges because interactions between bilayer lipids control cholesterol accessibility to toxins in a threshold-like fashion. Around these thresholds, the affinity of toxins for membrane cholesterol varies by >100-fold, generating highly cooperative lipid-dependent responses independently of protein-protein interactions. Such lipid-driven cooperativity may control the sensitivity of many cholesterol-dependent processes.     

Potentials of radio-frequency field gradient NMR microscopy in environmental science

An understanding of transport, flow, diffusivity and mass transfer processes is of central importance in many fields of environmental biotechnology such as biofilm, bioreactor and membrane engineering, soil and groundwater bioremediation, and wastewater treatment. Owing to its remarkable sensitivity to molecular displacements and to its noninvasive and nondestructive character, pulsed field gradient (PFG) nuclear magnetic resonance (NMR) can be a valuable tool for investigating such processes. In conventional NMR microscopy, spatial encoding is achieved by using static magnetic field gradients (B ₀ gradients). However, an interesting alternative is to use radio-frequency magnetic field gradients (RF or B ₁ gradients). Although the latter are less versatile than the former, RF field gradient microscopy is particularly suitable for dealing with heterogeneous systems such as porous media because of its quasi-immunity to background static magnetic field gradients arising from magnetic susceptibility inhomogeneities, unlike the B ₀ gradients microscopy. Here, we present an overview of basic principles and the main features of this technique, which is still relatively unused. Different examples of diffusion imaging illustrate the potentialities of the method in both micro-imaging and the measurement of global or local diffusion coefficients within membranes and at liquid–solid interfaces. These examples suggest that a number of environmental problems could benefit from this technique. Different future prospects of application of B ₁ gradient NMR microscopy in environmental biotechnology are considered. Journal of Industrial Microbiology & Biotechnology (2001) 26, 53–61. Received 09 February 2000/ Accepted in revised form 07 August 2000     

Biomimetic membrane systems to study cellular organization

During many cellular processes such as cell division, polarization and motility, the plasma membrane does not only represent a passive physical barrier, but also provides a highly dynamic platform for the interplay between lipids, membrane binding proteins and cytoskeletal elements. Even though many regulators of these interactions are known, their mutual interdependence appears to be highly complex and difficult to study in a living cell. Over the past few years, in vitro studies on membrane–cytoskeleton interactions using biomimetic membranes turned out to be extremely helpful to get better mechanistic insight into the dynamics of these processes. In this review, we discuss some of the recent developments using in vitro assays to dissect the role of the players involved: lipids in the membrane, proteins binding to membranes and proteins binding to membrane proteins. We also summarize advantages and disadvantages of supported lipid bilayers as model membrane.     

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.