Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55 % after 40 Ih incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling. (c) 2005 Elsevier B.V. All rights reserved.

Biomolecular engineering at interfaces

A broad review of technologically focused work concerning biomolecules at interfaces is presented. The emphasis is on developments in interfacial biomolecular engineering that may have a practical impact in bioanalysis, tissue engineering, emulsion processing or bioseparations. We also review methods for fabrication in an attempt to draw out those approaches that may be useful for product manufacture, and briefly review methods for analysing the resulting interfacial nanostructures. From this review we conclude that the generation of knowledge and-innovation at the nanoscale far exceeds our ability to translate this innovation into practical outcomes addressing a market need, and that significant technological challenges exist. A particular challenge in this translation is to understand how the structural properties of biomolecules control the assembled architecture, which in turn defines product performance, and how this relationship is affected by the chosen manufacturing route. This structure-architecture-process-performance (SAPP) interaction problem is the familiar laboratory scale-up challenge in disguise. A further challenge will be to interpret biomolecular self- and directed-assembly reactions using tools of chemical reaction engineering, enabling rigorous manufacturing optimization of self-assembly laboratory techniques. We conclude that many of the technological problems facing this field are addressable using tools of modem chemical and biomolecular engineering, in conjunction with knowledge and skills from the underpinning sciences. (c) 2005 Elsevier Ltd. All rights reserved.

The in-vitro bioactivity of mesoporous bioactive glasses

Ordered mesoporous bioactive glasses (MBGs) with different compositions were prepared by using nonionic block copolymer surfactants as structure-directing agents through an evaporation-induced self-assembly process. Their in-vitro bioactivities were studied in detail by electron microscopy, Fourier-transform infrared spectroscopy, and inductively coupled plasma (ICP) atomic emission spectroscopy. The ICP element analysis results were further calculated in terms of the total consumption of Ca and P, Delta[Ca]/Delta[P] ratios, and ionic activity product (IP) of hydroxyapatite. Through the above analysis, it is clear that MBGs show a different structure-bioactivity correlation compared to conventional sol-gel-derivcd BGs. The in vitro bioactivity of MBGs is dependent on the Si/Ca ratio in the network when the other material parameters such as the mesostructure and texture properties (pore size, pore volume) are controlled. MBG 80S15C with relatively lower calcium content exhibits the best in vitro bioactivity, in contrast to conventional sol-gel-derived BGs where usually higher calcium percentage BGs (e.g. 60S35C) show better bioactivity. Calcination temperature is another important factor that influences the in vitro bioactivity. According to our results, MBGs calcined at 973 K may possess the best in vitro bioactivity. The influences of the composition and calcination temperature upon bioactivity are explained in terms of the unique structures of MBGs. (c) 2006 Elsevier Ltd. All rights reserved.

Reversible active switching of the mechanical properties of a peptide film at a fluid-fluid interface

Designer peptides have recently been developed as building blocks for novel self-assembled materials with stimuli-responsive properties. To date, such materials have been based on self-assembly in bulk aqueous solution or at solid-fluid interfaces. We have designed a 21-residue peptide, AM1, as a stimuli-responsive surfactant that switches molecular architectures at a fluid-fluid interface in response to changes in bulk aqueous solution composition. In the presence of divalent zinc at neutral pH, the peptide forms a mechanically strong 'film state'. In the absence of metal ions or at acid pH, the peptide adsorbs to form a mobile 'detergent state'. The two interfacial states can be actively and reversibly switched. Switching between the two states by a change in pH or the addition of a chelating agent leads to rapid emulsion coalescence or foam collapse. This work introduces a new class of surfactants that offer an environmentally friendly approach to control the stability of interfaces in foams, emulsions and fluid-fluid interfaces more generally.

The economics of inclusion body processing

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.

The refolding of different alpha-fetoprotein variants

The effect of glycosylation on AFP foldability was investigated by parallel quantitative and qualitative analyses of the refolding of glycosylated and nonglycosylated AFP variants. Both variants were successfully refolded by dialysis from the denatured-reduced state, attaining comparable ``refolded peak'' profiles and refolding yields as determined by reversed-phase HPLC analysis. Both refolded variants also showed comparable spectroscopic fingerprints to each other and to their native counterparts, as determined by circular dichroism spectroscopy. Inclusion body-derived AFP was also readily refolded via dilution under the same redox conditions as dialysis refolding, showing comparable circular dichroism fingerprints as native nonglycosylated AFP. Quantitative analyses of inclusion body-derived AFP showed sensitivity of AFP aggregation to proteinaceous and nonproteinaceous inclusion body contaminants, where refolding yields increased with increasing AFP purity. All of the refolded AFP variants showed positive responses in ELISA that corresponded with the attainment of a bioactive conformation. Contrary to previous reports that the denaturation of cord serum AFP is an irreversible process, these results clearly show the reversibility of AFP denaturation when refolded under a redox-controlled environment, which promotes correct oxidative disulfide shuffling. The successful refolding of inclusion body-derived AFP suggests that fatty acid binding may not be required for the attainment of a rigid AFP tertiary structure, contrary to earlier studies. The overall results from this work demonstrate that foldability of the AFP molecule from its denatured-reduced state is independent of its starting source, the presence or absence of glycosylation and fatty acids, and the refolding method used (dialysis or dilution).

Generic Technique to Generate Large Branched DNA Complexes

The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.

Dilution versus dialysis - A quantitative study of the oxidative refolding of recombinant human alpha-fetoprotein

Alpha-fetoprotein (AFP) is a commercially important polypeptide with important diagnostic. physiological and immunomodulatory functions. Previous studies into the refolding of this macromolecule are contradictory. and variously suggest that AFP denaturation may be irreversible or that refolding may be achieved by reducing denaturant concentration through dilution but not dialysis. Importantly, these same previous studies do not provide quantitative metrics by which the Success of refolding, and the potential for bioprocess development. can be assessed. Moreover, these same studies do not optimize and control refolding redox potential - an important factor considering that AFP contains 32 cysteines which form 16 disulfide bonds. In this current study, a quantitative comparison of recombinant human AFP (rhAFP) refolding by dilution and dialysis is conducted under optimized redox conditions. rhAFP refolding yields were > 35% (dialysis refolding) and > 75% (dilution refolding) as assessed by RP-HPLC and ELISA, with structural Similarity to the native state confirmed by UV spectroscopy. Dialysis refolding yield was believed to be lower because the gradual reduction in denaturant concentration allowed extended conformational searching. enabling more time for undesirable interaction with other protein molecules and/or the dialysis membrane, leading to a Sub-optimal process outcome. Significant yield sensitivity to redox environment was also observed, emphasizing the importance of physicochemical optimization. This study demonstrates that very high refolding yields can be obtained, for a physiologically relevant protein, with optimized dilution refolding. The study also highlights the quantitative metrics and macromolecular physical spectroscopic 'fingerprints' required to facilitate transition from laboratory to process scale.

Mechanical properties of interfacial films formed by lysozyme self-assembly at the air-water interface

We present the first characterization of the mechanical properties of lysozyme films formed by self-assembly at the air-water interface using the Cambridge interfacial tensiometer (CIT), an apparatus capable of subjecting protein films to a much higher level of extensional strain than traditional dilatational techniques. CIT analysis, which is insensitive to surface pressure, provides a direct measure of the extensional stress-strain behavior of an interfacial film without the need to assume a mechanical model (e.g., viscoelastic), and without requiring difficult-to-test assumptions regarding low-strain material linearity. This testing method has revealed that the bulk solution pH from which assembly of an interfacial lysozyme film occurs influences the mechanical properties of the film more significantly than is suggested by the observed differences in elastic moduli or surface pressure. We have also identified a previously undescribed pH dependency in the effect of solution ionic strength on the mechanical strength of the lysozyme films formed at the air-water interface. Increasing solution ionic strength was found to increase lysozyme film strength when assembly occurred at pH 7, but it caused a decrease in film strength at pH 11, close to the pI of lysozyme. This result is discussed in terms of the significant contribution made to protein film strength by both electrostatic interactions and the hydrophobic effect. Washout experiments to remove protein from the bulk phase have shown that a small percentage of the interfacially adsorbed lysozyme molecules are reversibly adsorbed. Finally, the washout tests have probed the role played by additional adsorption to the fresh interface formed by the application of a large strain to the lysozyme film and have suggested the movement of reversibly bound lysozyme molecules from a subinterfacial layer to the interface.

Production of Enzymatically active ketosteroid isomerase following insoluble expression in Escherichia coli

Enzymatically active Delta(5)-3-ketosteroid isomerase (KSI) protein with a C-terminus his(6)-tag was produced following insoluble expression using Escherichia coli. A simple, integrated process was used to extract and purify the target protein. Chemical extraction was shown to be as effective as homogenization at releasing the inclusion body proteins from the bacteria] cells, with complete release taking less than 20 min. An expanded bed adsorption (EBA) column utilizing immobilized metal affinity chromatography (IMAC) was then used to purify the denatured KSI-(His(6)) protein directly from the chemical extract. This integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations. The integrated chemical extraction and EBA process achieved a very high purity (99%) and recovery (89%) of the KSI-(His(6)), with efficient utilization of the adsorbent matrix (9.74 mg KSI-(His(6))/mL adsorbent). Following purification the protein was refolded by dilution to obtain the biologically active protein. Seventy-nine percent of the expressed KSI-(His(6)) protein was recovered as enzymatically active protein with the described extraction, purification, and refolding process. In addition to demonstrating the operation of this intensified inclusion body process, a plate-based concentration assay detecting KSI-(His(6)) is validated. The intensified process in this work requires minimal optimization for recovering novel his-tagged proteins, and further improves the economic advantage of E. coli as a host organism. (c) 2006 Wiley Periodicals, Inc.