Dynamic behavior of adsorber membranes for protein recovery

In recent years there has been a considerable interest in developing membrane chromatography systems that function as a short, wide chromatographic column in which the adsorptive packing consists of one or more microporous membranes. This study reports the use of new adsorber membranes prepared by the incorporation of various types of ion exchange resins into an EVAL porous membrane for protein recovery. The obtained heterogeneous matrixes composed of solid particles surrounded by the polymeric film possess a good accessibility for the protein to the adsorptive sites. Furthermore, small particles can be embedded into porous polymeric structures without the disadvantages of classical chromatographic columns (high pressure drop, fouling and plugging sensitivity, low flow rate), but with the advantages of membrane technology (easy scale-up, low-pressure drop, high flow rate). The adsorptive membranes feature high static as well as dynamic protein adsorption capacities for operating flow rates ranging from 200 to 400 L h bar per m(2) and ionic strength of 20-200 mM. In a sequential desorption step by changing the pH and/or the ionic strength of the eluent, up to 90% protein recovery was obtained. Next to the separation, the mixed matrix adsorber membrane functions as a concentration medium since the protein can be concentrated up to 20-fold in the eluent. The adsorber membranes can be reused in multiple adsorption/desorption cycles with good adsorption performances.     

Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic.

Tat is an 86- to 104-amino-acid viral protein that activates human immunodeficiency virus type 1 expression, modifies several cellular functions, and causes neurotoxicity. Here, we determined the extent to which peptide fragments of human immunodeficiency virus type 1 BRU Tat1-86 produced neurotoxicity, increased levels of intracellular calcium ([Ca2+]i), and affected neuronal excitability. Tat31-61 but not Tat48-85 dose dependently increased cytotoxicity and levels of [Ca2+]i in cultured human fetal brain cells. Similarly, Tat31-61 but not Tat48-85 depolarized rat hippocampal CA1 neurons in slices of rat brain. The neurotoxicity and increases in [Ca2+]i could be significantly inhibited by non-N-methyl-D-aspartate excitatory amino acid receptor antagonists. Shorter 15-mer peptides which overlapped by 10 amino acids each and which represented the entire sequence of Tat1-86 failed to produce any measurable neurotoxicity. Although it remains to be determined if Tat acts directly on neurons and/or indirectly via glial cells, these findings do suggest that Tat neurotoxicity is conformationally dependent, that the active site resides within the first exon of Tat between residues 31 to 61, and that these effects are mediated at least in part by excitatory amino acid receptors.     

Mechanistic insights into the translocation of full length HIV-1 Tat across lipid membranes

The mechanism of how full length Tat (aa 1-86) crosses artificial lipid membranes was elucidated by means of fluorescence spectroscopy and fluorescence microscopy. It was shown that full length Tat (aa 1-86) neither forms pores in large unilamellar vesicles (LUVs) nor in giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In contrast, an N-terminally truncated Tat protein (aa 35-86) that lacks the structurally defined proline- and cysteine-rich region as well as the highly conserved tryptophan residue at position 11 generates pores in artificial POPC-membranes, through which a water-soluble dye up to a size of 10kDa can pass. By means of fluorescence microscopy, the transfer of fluorescently labeled full length Tat across POPC-bilayers was unambiguously visualized with a concomitant accumulation of the protein in the membrane interface. However, if the dye was attached to the protein, also pore formation was induced. The size of the pores was, however smaller than the protein size, i.e. the labeled protein with a mass of 11.6kDa passed the membrane, while a fluorescent dye with a mass of 10kDa was excluded from the vesicles' interior. The results demonstrate that pore formation is not the prime mechanism by which full length Tat crosses a membrane.     

Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure.

The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.     

Characteristics of HIV-Tat protein transduction domain

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains rich arginine and lysine residues, is responsible for the highly efficient transduction of protein through the plasma membrane. In addition, it can be secreted from infected cells and has the ability to enter neighboring cells. When the PTD of Tat is fused to proteins and exogenously added to cells, the fusion protein can cross plasma membranes. Recent reports indicate that the endogenously expressed Tat fusion protein can demonstrate biodistribution of several proteins. However, intercellular transport and protein transduction have not been observed in some studies. Therefore, this study examined the intercellular transport and protein transduction of the Tat protein. The results showed no evidence of intercellular transport (biodistribution) in a cell culture. Instead, the Tat fusion peptides were found to have a significant effect on the transduction and intercellular localization properties. This suggests that the HIV-1 PTD passes through the plasma membrane in one direction.     

Characterization and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system

Proteins bearing a signal peptide with a consensus twin-arginine motif are translocated via the Tat pathway, a multiprotein system consisting minimally of the integral inner membrane proteins TatA, TatB, and TatC. On a molar basis, TatA is the major pathway component. Here we show that TatA can be purified independently of the other Tat proteins as a 460 kDa homooligomeric complex. Homooligomer formation requires the amino-terminal membrane-anchoring domain of TatA. According to circular dichroism spectroscopy, approximately half of the TatA polypeptide forms alpha-helical secondary structure in both detergent solution and proteoliposomes. An expressed construct without the transmembrane segment is largely unstructured in aqueous solution but is able to insert into phospholipid monolayers and interacts with membrane bilayers. Protease accessibility experiments indicate that the extramembranous region of TatA is located at the cytoplasmic face of the cell membrane.     

Membrane interactions and self-association of the TatA and TatB components of the twin-arginine translocation pathway

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin-arginine signal peptides. The essential Tat pathway components TatA, TatB and TatC are shown to be integral membrane proteins. Upon removal of the predicted N-terminal transmembrane helix TatA becomes a water-soluble protein. In contrast the homologous TatB protein retains weak peripheral interactions with the cytoplasmic membrane when the analogous helix is deleted. Chemical crosslinking studies indicate that TatA forms at least homotrimers, and TatB minimally homodimers, in the native membrane environment. The presence of such homo-oligomeric interactions is supported by size exclusion chromatography.     

Chromogranin, an integral membrane protein

Chromogranin is the major soluble protein of the adrenal medulla chromaffin granule and is secreted upon nervous stimulation. Using antisera to pure chromogranin in immunoblotting procedures, we show that chromogranin is the major integral membrane protein as well. Extraction of chromaffin granule membranes with low salt, high salt, chelating agents, or calcium-containing solutions does not remove the chromogranin from the membranes. The membrane form of chromogranin can be purified on a C-18 semi-preparative column using high pressure liquid chromatography. Amino-terminal sequence data indicate that the membrane and soluble forms of chromogranin are identical or very similar.     

The Chloroplast Twin Arginine Transport (Tat) Component,Tha4, Undergoes Conformational Changes Leading to Tat Protein Transport

Twin arginine transport (Tat) systems transport folded proteins using proton-motive force as sole energy source. The thylakoid Tat system comprises three membrane components. A complex composed of cpTatC and Hcf106 is the twin arginine signal peptide receptor. Signal peptide binding triggers assembly of Tha4 for the translocation step. Tha4 is thought to serve as the protein-conducting element, and the topology it adopts during transport produces the transmembrane passageway. We analyzed Tha4 topology and conformation in actively transporting translocases and compared that with Tha4 in nontransporting membranes. Using cysteine accessibility labeling techniques and diagnostic protease protection assays, we confirm an overall N_OUT-C_IN topology for Tha4 that is maintained under transport conditions. Significantly, the amphipathic helix (APH) and C-tail exhibited substantial changes in accessibility when actively engaged in protein transport. Compared with resting state, cysteines within the APH became less accessible to stromally applied modifying reagent. The APH proximal C-tail, although still accessible to Cys-directed reagents, was much less accessible to protease. We attribute these changes in accessibility to indicate the Tha4 conformation that is adopted in the translocase primed for translocation. We propose that in the primed translocase, the APH partitions more extensively and uniformly into the membrane interface and the C-tails pack closer together in a mesh-like network. Implications for the mode by which the substrate protein crosses the bilayer are discussed.     

A mechanistic investigation of cell-penetrating Tat peptides with supported lipid membranes

The multifarious Tat peptide derived from the HIV-1 virus exhibits antimicrobial activity. In this article, we use Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to investigate the mechanisms of action of Tat (44-57) and Tat (49-57) on bacterial-mimetic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DMPG) membranes. The results reveal that both peptides disrupt DMPC/DMPG membranes via a surface-active (carpet-like) mechanism. The magnitude of this disruption is dependent on both membrane and peptide properties. Firstly, less disruption was observed on the more negatively charged membranes. Secondly, less disruption was observed for the longer and slightly more hydrophobic Tat (44-57) peptide. As a comparison, the behaviour of the two Tat peptides on mammalian-mimetic DMPC/cholesterol membranes was investigated. Consistent with the literature no membrane disruption was observed. These results suggest that both electrostatic and hydrophobic interactions, as well as peptide geometry, determine the antimicrobial activity of Tat. This should guide the development of more potent Tat antibiotics.     

Protein binding to polymer brush,based on ion-exchange,hydrophobic, and affinity interactions

The major limitations associated with conventional packed bed chromatography for protein separation and purification can be overcome by using adsorptive microporous membranes as chromatographic media. Microporous membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate diffusion limitations. As a result, higher throughput and shorter processing times are possible using these membrane systems. In this paper, we review the current state of development in the area of attaching functionalized polymer brushes onto a microporous membrane to form a novel chromatographic medium for protein separation and purification. The functionalized polymer brushes were appended onto the pore surface of a microporous hollow-fiber membrane uniformly across the membrane thickness by radiation-induced graft polymerization and subsequent chemical modifications. We review various applications of this adsorptive membrane chromatography by focusing on polymer brushes bearing ion-exchange, hydrophobic and affinity groups. Proteins were captured in multilayers by the ion-exchange group-containing polymer brushes due to the formation of a three-dimensional space for protein binding via the electrostatic repulsion of the polymer brushes. In contrast, proteins were captured in a monolayer at most by the polymer brushes containing hydrophobic or affinity ligands. By permeating a protein solution through the pores rimmed by the polymer brushes, an ideal capturing rate of the proteins with a negligible diffusional mass-transfer resistance was achieved by the functionalized polymer brushes, based on ion-exchange, hydrophobic, and affinity interactions.     

Membrane chromatography: preparation and applications to protein separation

As a result of the convective flow of solutes through porous membranes, membrane chromatography has a higher capture efficiency and a higher productivity than column chromatography and shows most promising industrial applications for the recovery, isolation, and purification of proteins and enzymes. This paper presents a comprehensive review of the methods for preparation of adsorptive membranes (such as surface modification, in situ copolymerization, direct formation from hydrophilic materials, and functionalized particulate-entrapped membranes) and deals particularly with novel macroporous chitin and chitosan membranes for protein separations developed by the authors.     

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.     

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein.

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.     

Size-exclusion reaction chromatography (SERC): a new technique for protein PEGylation

A new, widely applicable process that combines reaction and separation in a single unit operation is described. The process, size-exclusion reaction chromatography (SERC), simultaneously allows control of the extent of reactions in which molecular size is altered and the separation of products and reactants. In SERC, a moving reaction zone is formed by injection of reactants onto a size-exclusion chromatography column. Reactants and products are partitioned differently within the mobile phase, resulting in different linear flow rates through the column. The products are therefore removed selectively from the reaction zone, minimizing their residence time in the reaction zone and allowing their separation in the downstream section of the column. For reactions such as protein PEGylation, in which successive addition of PEG groups to the protein results in significant molecular size increases, SERC potentially offers a method by which a dominant final PEGylated protein size can be produced at high yield. The SERC PEGylation of two model proteins, alpha-lactalbumin and beta-lactoglobulin, is demonstrated and results show that simultaneous reaction and separation was obtained.     

Membrane targeting of a folded and cofactor-containing protein.

Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms. In bacteria, the twin arginine translocation (Tat) system can transport folded proteins. Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli. In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E. coli by a process requiring ATP when the Tat substrate was properly folded. During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state. Membrane targeting did not require a twin arginine motif and known Tat system components. On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane.     

Intracellular human immunodeficiency virus Tat expression in astrocytes promotes astrocyte survival but induces potent neurotoxicity at distant sites via axonal transport

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial-neuronal relationships is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. We thus initially transfected astrocytes with HIV proviral DNA and confirmed Tat production in these cells. Subsequently, using stably Tat-producing asytocyte cell lines, we observed that Tat promoted astrocyte survival by causing a prominent antioxidant effect and resistance to cell injury in these cells. Tat was released extracellularly where it could be taken up by other cells. Tat remained functionally active following uptake and caused long terminal repeat (LTR) transactivation in lymphocytic and astrocytic cell lines. Tat released from astrocytes caused mitochondrial dysfunction, trimming of neurites, and cell death in neurons. Tat neurotoxicity was attenuated by anti-Tat antibodies, kynurenate or heparan sulfate. The neurotoxic effects of Tat were caused at concentrations lower than that needed to cause LTR transactivation. When Tat-expressing cells were injected into the rat dentate gyrus, Tat was taken up by granule cells and transported along neuronal pathways to the CA3 region where it caused glial cell activation and neurotoxicity. The arginine-rich domain of Tat was essential for both the LTR transactivation and the neurotoxic properties of Tat. Thus HIV-Tat is a potent neurotoxin that may act at distant sites while at the same time it assures its production by preventing cell death in astrocytes where it is produced.     

Proteins and glycoproteins of the erythrocyte membrane

Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by acrylamide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).