Owen R.T. Thomas

Protein purification using magnetic adsorbent particles.

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.g. fermentation broths, cell disruptates, plasma, milk, whey and plant extracts) simply by binding them on magnetic adsorbents before application of a magnetic field. By using magnetic separation in this way, the several stages of sample pretreatment (especially centrifugation, filtration and membrane separation) that are normally necessary to condition an extract before its application on packed bed chromatography columns, may be eliminated. Magnetic separations are fast, gentle, scaleable, easily automated, can achieve separations that would be impossible or impractical to achieve by other techniques, and have demonstrated credibility in a wide range of disciplines, including minerals processing, wastewater treatment, molecular biology, cell sorting and clinical diagnostics. However, despite the highly attractive qualities of magnetic methods on a process scale, with the exception of wastewater treatment, few attempts to scale up magnetic operations in biotechnology have been reported thus far. The purpose of this review is to summarise the current state of development of protein separation using magnetic adsorbent particles and identify the obstacles that must be overcome if protein purification with magnetic adsorbent particles is to find its way into industrial practice.

Non-porous magnetic chelator supports for protein recovery by immobilised metal affinity adsorption

Abstract Micron-sized non-porous magnetic adsorbents derivatized with the metal chelating agent, iminodiacetic acid (IDA), have been prepared for the selective recovery of proteins. Four preparative routes employing epoxide activation chemistry were investigated to introduce IDA onto the surface of polyglutaraldehyde-coated particles. The presence of surface bound IDA was demonstrated by the selective binding of Cu 2 + and by the behaviour of Cu 2 + -charged and uncharged supports towards native haem proteins known to bind porous polymer-based Cu 2 + -IDA adsorbents. The simplest and most direct procedure was developed further. Supports prepared by this method were optimised with respect to ligand density and specific binding capacity. These coating and derivatization methods resulted in supports with a high level of substitution and low non-specific binding while retaining a high effective surface area for binding of the target protein (> 200 mg g −1 ). The resulting magnetic chelator supports possess excellent long term storage stability.

High gradient magnetic separation versus expanded bed adsorption: a first principle comparison

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the ‘loaded’ adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h−1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h−1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h−1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.

Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.

Demonstration of a strategy for product purification by high-gradient magnetic fishing: recovery of superoxide dismutase from unconditioned whey.

A systematic approach for the design of a bioproduct recovery process employing magnetic supports and the technique of high‐gradient magnetic fishing (HGMF) is described. The approach is illustrated for the separation of superoxide dismutase (SOD), an antioxidant protein present in low concentrations (ca. 0.15–0.6 mg L−1) in whey. The first part of the process design consisted of ligand screening in which metal chelate supports charged with copper(II) ions were found to be the most suitable. The second stage involved systematic and sequential optimization of conditions for the following steps: product adsorption, support washing, and product elution. Next, the capacity of a novel high‐gradient magnetic separator (designed for biotechnological applications) for trapping and holding magnetic supports was determined. Finally, all of the above elements were assembled to deliver a HGMF process for the isolation of SOD from crude sweet whey, which consisted of (i) binding SOD using Cu2+‐charged magnetic metal chelator particles in a batch reactor with whey; (ii) recovery of the “SOD‐loaded” supports by high‐gradient magnetic separation (HGMS); (iii) washing out loosely bound and entrained proteins and solids; (iv) elution of the target protein; and (v) recovery of the eluted supports from the HGMF rig. Efficient recovery of SOD was demonstrated at ∼50‐fold increased scale (cf. magnetic rack studies) in three separate HGMF experiments, and in the best of these (run 3) an SOD yield of >85% and purification factor of ∼21 were obtained.

Characterisation of non-porous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E. coli extract.

The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents. Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E. coli extracts in a single step. Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose. The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale. By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.

SELECTIVE FLOCCULATION OF CELLULAR CONTAMINANTS FROM SOLUBLE-PROTEINS USING POLYETHYLENEIMINE - A STUDY OF SEVERAL ORGANISMS AND POLYMER MOLECULAR-WEIGHTS

Saccharomyces cerevisiae, Escherichia coli, and Pseudomonas putida homogenates were treated with polyethyleneimine (PEI) of various molecular weights and concentrations, and the removal of cellular contaminants noted. The optimum doses were determined and the effects of ionic strength observed. Although there was little variation between the abilities of the various PEIs to remove cellular contaminants, the possibility of floc redissolution at higher polymer concentrations occurred more with increased molecular weight of the polymer. Increasing the ionic strength decreased the occurrence of floc redissolution. Both E. coli and P. putida required a higher concentration of PEI than S. cerevisiae to achieve significant removal of cellular contaminants. Loss of soluble protein by absorption to flocs with E. coli was influenced strongly by ionic strength.

The processing of a plasmid-based gene from E. Coli. Primary recovery by filtration

Abstract We describe the primary recovery of plasmid DNA from alkaline lysis mixtures using a nutsche filter operated under pressure. Six different filter cloths constructed of polypropylene, polyester and stainless steel were tested, with pore sizes ranging from 5–160␣μm. Both pore size and the material of the filter membranes employed in filtration experiments exerted considerable impact on the purity and yield of the plasmid DNA. The greatest degree of solids extrusion, shearing of chromosomal DNA and subsequent contamination of the filtrate was observed with the 160␣μm polyester filter. The best compromise was obtained with a 5␣μm polypropylene cloth. For an alkaline lysis mixture containing 101␣g wet weight solids per litre, filtration through this cloth proceeded at an average rate of 22.5␣cm␣h−1. Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold purification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoating with diatomaceous earths of different permeabilities (0.07–1.2␣darcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and < 1% of the original protein remaining in the filtrate. However, the plasmid yield was only 30%, the processing rate was markedly reduced (8.2␣cm␣h−1), and some losses of plasmid DNA, due to adsorption on to the diatomaceous earth, were also observed (5.7%).

Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: A novel strategy exploiting disulfide assisted protein folding

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC‐I) heavy‐chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy‐chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1) correct folding can only take place in combined presence of β2‐microglobulin and a binding peptide; and (2) optimal in vitro conditions for disulfide bond formation (∼pH 8) and peptide binding (∼pH 6.6) are far from complementary. Here we present a two‐step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy‐chain molecules with correct disulfide bonding are formed under non‐reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond–assisted folding in the presence of β2‐microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.

Studies related to antibody fragment (Fab) production in Escherichia coli W3110 fed‐batch fermentation processes using multiparameter flow cytometry

Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti‐lysozyme Fab fragment under the control of the lac‐based expression system, were taken at various stages from fed‐batch fermentation processes and stained with a mixture of bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX). Where appropriate, measurements of dissolved oxygen tension (DOT), OD600nm and Fab concentration were made. Depending on time of induction the maximum amount of Fab accumulating in the supernatant varied quite markedly from 1 to 4 μg ml−1 as did subsequent cell physiological state with respect to PI/BOX staining with a concomitant drop in maximum biomass concentration. Depending on point of induction a fourfold increase in Fab production could be achieved accompanied by a ∼50% drop in maximum biomass concentration but with a higher proportion of viable cells as measured by multiparameter flow cytometry.