Protein renaturation with simultaneous purification by protein folding liquid chromatography: recent developments

Protein folding liquid chromatography (PFLC) is a powerful tool for protein refolding with simultaneous purification. We review its recent progress in liquid chromatography and molecular biology, primarily involving the validation of PFLC refolding of proteins containing multiple disulphide bonds, the application of mixed-mode chromatography, PFLC in molecular biology. Representative examples are described.     

Protein adsorption modalities on polyelectrolyte multilayers

Protein adsorption on polyelectrolyte multilayers (PEMUs) was evaluated using a combination of synthetic polyelectrolytes and proteins, including serum albumin, fibrinogen, and lysozyme. Variables such as surface and protein charge, polymer hydrophobicity, and hydrophilic repulsion were introduced to probe interaction mechanisms. Quantitative analysis with reflectance Fourier transform infrared spectroscopy, optical waveguiding, and UV-vis absorption, together with qualitative information from atomic force microscopy, provided a coordinated picture for what drives protein adsorption and how the molecules are disposed on the multilayer surface. It was found that multilayers bearing a particular surface charge sorbed biomolecules if they were of opposite charge, yielding significant loadings within the bulk PEMU. Adsorption of like-charged proteins, as surface aggregates, occurred to a much lower extent, driven by nonelectrostatic forces. A diblock copolymer comprising a hydrophilic poly(ethylene oxide) block was capable of further minimizing protein adsorption as a result of hydrophilic repulsion, although none of the surfaces tested defeated protein adsorption completely. However, poly(acrylic acid) homopolymer was quite effective in this respect. A composition gradient, formed during multilayer buildup, induced a gradient in hydrophilicity through the PEMU, which is an efficient and economical method of creating a protein-resistant surface.     

Protein engineering to optimize recombinant protein purification

Genetic approaches have been used to facilitate purification of recombinant proteins, on both a large and a small scale. Based on developments in three different areas: (i) affinity chromatography; (ii) specific cleavage of fusion proteins and (iii) secretion of fusion proteins, a coupled expression/secretion system was designed. It was further improved by protein engineering. Using a synthetic DNA fragment, encoding two IgG-binding domains derived from staphylococcal protein A, gene products were secreted to the culture medium of Escherichia coli and purified with a one-step affinity procedure. The system has been used for large-scale production of biologically active human peptide hormones, to generate peptides for antibody production and to immobilize proteins on solid supports.     

Profiling charge complementarity and selectivity for binding at the protein surface

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.     

Preparative purification of a recombinant protein by hydrophobic interaction chromatography: modulation of selectivity by the use of chaotropic additives

Development and implementation of a chaotropic wash step following protein loading on a hydrophobic interaction chromatographic (HIC) column is described for the purification of a recombinant protein. Various agents that reduce protein affinity in hydrophobic interaction chromatographic systems were screened for their utility in a wash step following protein loading on a Phenyl Fast Flow Sepharose HIC column. A combination of sodium thiocyanate, glycerol, and urea was selected as a suitable additive for the wash buffer that selectively eluted most of the major impurities present in the feed stream. Eluate purity, as monitored by reversed-phase chromatography and SDS-PAGE, was significantly increased by incorporation of this wash step in the purification process. Incorporation of this wash step on HIC enabled a reduction in the overall number of chromatographic steps in the downstream purification process for this recombinant protein, resulting in improved process yields and significant economic advantages.The effect of varying concentrations of each of the three wash additives on yield was studied. While the step yield decreased with an increase in concentration for urea and sodium thiocyanate, an optimum was observed with respect to glycerol concentration. The preferential interaction theory is employed to explain this effect.     

Protein purification by Off-Gel electrophoresis

A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.     

Protein purification by affinity precipitation

Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Competing interactions for antimicrobial selectivity based on charge complementarity

Antimicrobial peptides (AMPs) are an evolutionary conserved component of the innate immune system and possible templates for the development of new antibiotics. An important property of antimicrobial peptides is their ability to discriminate bacterial from eucaryotic cells which is attributed to the difference in lipid composition of the outer leaflet of the plasma membrane between the two types of cells. Whereas eucaryotic cells usually expose zwitterionic lipids, procaryotic cells expose also anionic lipids which bind the cationic antimicrobial peptides electrostatically. An example is the antimicrobial peptide NK-2 which is highly cationic and favors binding to anionic membranes. In the present study, the difference in binding affinity of NK-2 for palmitoyl-oleoyl-phosphatidyl-glycerol (POPG) and palmitoyl-oleoyl-phosphatidyl-choline (POPC) is studied using molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration, by computing the change in free energy and its components upon the transfer of NK-2 from POPC to POPG. The transfer is indeed found to be highly favorable. Interestingly, the favorable contribution from the electrostatic interaction between the peptide and the anionic lipids is overcompensated by an unfavorable contribution from the change in lipid-cation interactions due to the release of counterions from the lipids. The increase in entropy due to the release of the cations is compensated by other entropic components. The largest favorable contribution arises from the solvation of the counterions. Overall the interaction between NK-2 and POPG is not determined by a single driving force but a subtle balance of competing interactions.     

Free-flow electrophoresis for purification of plant mitochondria by surface charge

Sample purity is the key for a successful in-depth analysis of any given subcellular proteome. The suitability of free-flow electrophoresis to assist conventional, centrifugation-based techniques in the preparation of plant mitochondria from green and non-green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.     

The influence of purification and protein heterogeneity on the crystallization of p-hydroxybenzoate hydroxylase

The structure of the enzyme p-hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm [Wierenga et al. (1979) J. Mol. Biol. 131, 53-73] with crystals belonging to space group C222(1). Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained. We have thoroughly investigated this problem and found that Cibacron-blue-purified enzyme appears to be heterogeneous with respect to aggregation state and Cys-116 oxidation. Most importantly, it could be firmly established that C222(1) crystals can only be grown from purely dimeric p-hydroxybenzoate hydroxylase possessing an intact SH group. Ion-exchange chromatography on DEAE-Sepharose can successfully remove those forms of the enzyme which impede successful crystallization. Sulfite and dithiothreitol improve crystallization by dissociating the enzyme oligomers into dimers; sulfite especially gives excellent results.     

Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.     

Effects of polyelectrolyte chain stiffness, charge mobility, and charge sequences on binding to proteins and micelles

The binding affinities of polyanions for bovine serum albumin in NaCl solutions from I = 0.01-0.6 M, were evaluated on the basis of the pH at the point of incipient binding, converting each such pH(c) value into a critical protein charge Zc. Analogous values of critical charge for mixed micelles were obtained as the cationic surfactant mole fraction Yc. The data were well fitted as Yc or Zc = KI a, and values of K and a were considered as a function of normalized polymer charge densities (tau), charge mobility, and chain stiffness. Binding increased with chain flexibility and charge mobility, as expected from simulations and theory. Complex effects of tau were related to intrapolyanion repulsions within micelle-bound loops (seen in the simulations) or negative protein domain-polyanion repulsions. The linearity of Zc with radicalI at I < 0.3 M was explained by using protein electrostatic images, showing that Zc at I < 0.3 M depends on a single positive "patch"; the appearance of multiple positive domains I > 0.3 M (lower pH(c)) disrupts this simple behavior.     

Protein phosphorylation in Escherichia coli and purification of a protein kinase

More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography. The protein species and the level of phosphorylation varied depending on the cell growth phase. With [gamma-32P]ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. Both serine and threonine were the major phosphate acceptors in whole cell lysates. Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E. coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity. The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides. We also isolated a 100K protein with self-phosphorylation activity.     

Species selectivity in poxviral complement regulators is dictated by the charge reversal in the central complement control protein modules

Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.     

ZZ made EZ: influence of inhibitor configuration on enzyme selectivity

Selectivity of drug targeting is necessary in order to forestall undesired side-effects. Here, we examine the structural grounds for the configuration-dependent selectivity of 2,7-bis(4-amidinobenzylidene)-cycloheptan-1-one (1) for factor Xa and trypsin: Previous studies showed that factor Xa is preferentially inhibited by the (Z,Z) configuration isomer of (1), whilst trypsin binds equally well to both (E,Z) and (Z,Z) forms. Using engineered trypsin variants, we find similar overall binding modes for the (E,Z) and (Z,Z) isomers. Minor changes in van der Waals' contacts to Tyr99 (Leu in trypsin) explain the differential inhibition of factor Xa. We note differences in the experimental electron densities observed from co-crystallisation and soaking experiments: while the co-crystallisation of (1) with variants containing Tyr99 (Leu99) reveal the exclusive presence of the (Z,Z) ((E,Z)) configurations respectively, soaking experiments with either variant result in mixtures of (E,Z), (Z,Z) and (E,E). This discrepancy arises presumably from differences in the spatial (packing considerations) or chemical (crystallisation conditions) microenvironments. The results presented here represent an extreme example of the problems that face structure-based drug design, in particular the dangers inherent in relying on a single crystal structure for interpreting protein-ligand interactions.     

The influence of anisotropy on brain injury prediction

Traumatic Brain Injury (TBI) occurs when a mechanical insult produces damage to the brain and disrupts its normal function. Numerical head models are often used as tools to analyze TBIs and to measure injury based on mechanical parameters. However, the reliability of such models depends on the incorporation of an appropriate level of structural detail and accurate representation of the material behavior. Since recent studies have shown that several brain regions are characterized by a marked anisotropy, constitutive equations should account for the orientation-dependence within the brain. Nevertheless, in most of the current models brain tissue is considered as completely isotropic. To study the influence of the anisotropy on the mechanical response of the brain, a head model that incorporates the orientation of neural fibers is used and compared with a fully isotropic model. A simulation of a concussive impact based on a sport accident illustrates that significantly lowered strains in the axonal direction as well as increased maximum principal strains are detected for anisotropic regions of the brain. Thus, the orientation-dependence strongly affects the response of the brain tissue. When anisotropy of the whole brain is taken into account, deformation spreads out and white matter is particularly affected. The introduction of local axonal orientations and fiber distribution into the material model is crucial to reliably address the strains occurring during an impact and should be considered in numerical head models for potentially more accurate predictions of brain injury.