Detection and prevention of protein aggregation before,during, and after purification

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.     

Separating full-length protein from aggregating proteolytic products using filter flow-through purification

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Isolation of Three Crystalline Proteins from Beef Liver After Partial Purification by Acetone Fractionation

The isolation of three proteins in crystalline form from ground beef liver is described. These proteins are FTBL protein (Arch. Biochem. Biophys. 188, 251–265 (1978), crotonase, and catalase. Crotonase is isolated by crystallization from a 32 acetone extract of the ground liver. FTBL protein and catalase can subsequently be isolated from the same extract. For optimal yield and ease of isolation, FTBL protein is isolated from a 46.5% acetone extract from which catalase can subsequently be crystallized by dialysis.

The isolation of FTBL protein as well as the isolation of catalase involves a preliminary fractional precipitation and solution before crystallization can be achieved. Isopropanol can be substituted for acetone in the isolation of the above three proteins and in the case of catalase, results in an exceptionally high yield.

Methods for the recrystallization of the proteins are presented and the role of organic solvents in recrystallization is discussed.     

The thermodynamics of water-protein interactions

Information about the effects of water on protein structure and function can be obtained from studies on freeze dried protein powders of varying water content. Sorption isotherms of water on proteins can be used to obtain thermodynamic quantities for water-protein interactions. Since such isotherms show hysteresis, there is doubt in regard to their interpretation.General expressions for the thermodynamic quantities of sorption are derived. If isotherms represent data at equilibrium, it is possible to calculate these thermodynamic quantities.There are two types of hysteresis, non-equilibrium hysteresis and equilibrium hysteresis. Absorption and desorption isotherms can show equilibrium hysteresis if different protein conformations, which are only slowly interconvertible, can be present. In this case valid thermodynamic quantities can be obtained. Experimental tests for equilibrium hysteresis are presented. More experiments are needed before definite conclusions can be drawn in regard to isotherms in the literature.If the protein conformation in a protein powder is similar to the protein conformation in aqueous solution, equilibrium data obtained from sorption isotherms can be used to approximate thermodynamic quantities for the interaction of water with proteins in aqueous solution. Examination of what experimental evidence is available indicates that the protein in powders prepared by desorption of water should have a conformation similar to that in solution. Further study of such samples will help to clarify the thermodynamics of water-protein interactions in aqueous solution.     

A database and tools for 3-D protein structure comparison and alignment using the Combinatorial Extension (CE) algorithm

The database reported here is derived using the Combinatorial Extension (CE) algorithm which compares pairs of protein polypeptide chains and provides a list of structurally similar proteins along with their structure alignments. Using CE, structure-structure alignments can provide insights into biological function. When a protein of known function is shown to be structurally similar to a protein of unknown function, a relationship might be inferred; a relationship not necessarily detectable from sequence comparison alone. Establishing structure-structure relationships in this way is of great importance as we enter an era of structural genomics where there is a likelihood of an increasing number of structures with unknown functions being determined. Thus the CE database is an example of a useful tool in the annotation of protein structures of unknown function. Comparisons can be performed on the complete PDB or on a structurally representative subset of proteins. The source protein(s) can be from the PDB (updated monthly) or uploaded by the user. CE provides sequence alignments resulting from structural alignments and Cartesian coordinates for the aligned structures, which may be analyzed using the supplied Compare3D Java applet, or downloaded for further local analysis. Searches can be run from the CE web site, http://cl.sdsc.edu/ce.html, or the database and software downloaded from the site for local use.     

Strategy for the Use of Affinity Grids to Prepare Non-His-Tagged Macromolecular Complexes for Single-Particle Electron Microscopy

Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized nickel-nitrilotriacetic acid lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here, we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II, still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-Å-resolution density map by single-particle cryo-EM.     

Determination of protein oligomeric structure from small‐angle X‐ray scattering

Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.     

Building proteins from C alpha coordinates using the dihedral probability grid Monte Carlo method.

Dihedral probability grid Monte Carlo (DPG-MC) is a general-purpose method of conformational sampling that can be applied to many problems in peptide and protein modeling. Here we present the DPG-MC method and apply it to predicting complete protein structures from C alpha coordinates. This is useful in such endeavors as homology modeling, protein structure prediction from lattice simulations, or fitting protein structures to X-ray crystallographic data. It also serves as an example of how DPG-MC can be applied to systems with geometric constraints. The conformational propensities for individual residues are used to guide conformational searches as the protein is built from the amino-terminus to the carboxyl-terminus. Results for a number of proteins show that both the backbone and side chain can be accurately modeled using DPG-MC. Backbone atoms are generally predicted with RMS errors of about 0.5 A (compared to X-ray crystal structure coordinates) and all atoms are predicted to an RMS error of 1.7 A or better.     

pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.

We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.     

Ruleminer: a knowledge system for supporting high-throughput protein function annotations

In this paper, we present RuleMiner, a knowledge system to facilitate a seamless integration of multi-sequence analysis tools and define profile-based rules for supporting high-throughput protein function annotations. This system consists of three essential components, Protein Function Groups (PFGs), PFG profiles and rules. The PFGs, established from an integrated analysis of current knowledge of protein functions from Swiss-Prot database and protein family-based sequence classifications, cover all possible cellular functions available in the database. The PFG profiles illustrate detailed protein features in the PFGs as in sequence conservations, the occurrences of sequence-based motifs, domains and species distributions. The rules, extracted from the PFG profiles, describe the clear relationships between these PFGs and all possible features. As a result, the RuleMiner is able to provide an enhanced capability for protein function analysis, such as results from the integrated sequence analysis tools for given proteins can be comparatively analyzed due to the clear feature-PFG relationships. Also, much needed guidance is readily available for such analysis. If the rules describe one-to-one (unique) relationships between the protein features and the PFGs, then these features can be utilized as unique functional identifiers and cellular functions of unknown proteins can be reliably determined. Otherwise, additional information has to be provided.     

Proteolysis of protein C in pooled normal plasma and purified protein C by activated protein C (APC)

Protein C is a vitamin-K dependent zymogen of the anti-coagulant serine protease activated protein C (APC). In this paper, we report four lines of evidence that APC can activate protein C in pooled normal plasma, and purified protein C. First, the addition of APC to protein C-deficient plasma supplemented with protein C produces a prolongation of the clotting time of plasma that is proportional to the amount of protein C. This behavior was observed with APC from the Chromogenix APC resistance kit (Dia Pharm, Franklin, OH, USA) and from APC derived from the thrombin activation of human protein C (Enzyme Research Laboratories, South Bend, IN, USA). Secondly, using immunoblotting after gel electrophoresis, the disappearance of epitopes for monoclonal antibodies that recognize protein C but not APC indicates a time course for the activation by APC of protein C in pooled normal plasma and protein C purified from plasma. Thirdly, the same time course for the disappearance of protein C specific epitope can be followed using ELISA. Finally, protein C can be activated by APC as indicated by the increase in APC specific synthetic substrate Tryp-Arg-Arg-p nitroaniline hydrolysis. Kinetic data indicate a value of 4.7+/-0.4 mM(-1) s(-1) for the activation of protein C by APC under physiological conditions and in the presence of calcium. These observations document that APC must function not only in the inactivation of activated factors V and VIII, but also in the activation of protein C. This additional action of APC may be important to consider more broadly because of APC in the treatment of sepsis.     

Effect of invertase on the batch foam fractionation of bromelain

Foam fractionation can be used to enrich a hydrophobic protein such as bromelain from an aerated dilute protein solution because the protein foams. On the other hand, a protein such as invertase, which is hydrophilic, is not likely to foam under similar aerated conditions. While a foam fractionation process may not be approapriate for recovering a hydrophilic protein alone, it is of interest to see how that non-foaming protein affects the foaming protein when the two are together in a mixture. The bromelain enrichment, activity and mass recovery were observed as a function of the solution pH in order to explore how invertase can affect the recovery of bromelain in a foam fractionation process.     

Purification of the gene 0.3 protein of bacteriophage T7, an inhibitor of the DNA restriction system of Escherichia coli

The gene 0.3 protein of bacteriophage T7 prevents the DNA restriction system of EScherichia coli from interfering with T7 infection. A mutant strain of T7 that greatly overproduces the 0.3 protein has been constructed and used for purification of this protein. The 0.3 protein ws found to be extremely acidic and can be separated from virtually all other proteins of the infected cell by chromatography on DEAE-cellulose. Residual contaminating proteins and nucleic acids can be removed by gel filtration, but an even simpler final purification is possible, because under appropriate conditions the 0.3 protein is soluble in high concentrations of ethanol. Thus, a simple, essentially two-step purification can produce about 50 mg of pure 0.3 protein from 30 liters of culture. The purified protein appears to be a dimer of identical subunits. AS expected from its known function during infection, the purified 0.3 protein inhibits the nuclease and ATPase activities of partially purified Eco B, the DNA restriction enzyme of E. coli B, but it does not interfere with several different type II endonucleases tested. The inhibition of Eco B appears to require stoichiometric rather than catalytic amounts of 0.3 protein.     

An improved protein binding assay for guanosine-3',5'-monophosphate using a binding protein from the pupa of the silkmoth, Bombyx mori L.

A modification of the protein binding assay for cyclic guanosine-3',5'-monophosphate (cyclic GMP) is described that is more sensitive and less subject to interference by cyclic AMP than are previously published protein binding methods. The assay employs a purified binding protein from the fat body of the pupa of the common silkmoth, Bombyx mori. The dissociation constant of the binding protein for cyclic GMP is 4.3 nM. A protein kinase modulator protein isolated from the same species increases the binding affinity and capacity of the cyclic GMP binding protein and can be used to advantage in the assay for cyclic GMP. As little as 0.1 pmoles of cyclic GMP can be detected by this procedure. Changes in the level of cyclic GMP in the frog heart during the cardiac cycle were determined by means of the new assay.     

Biosensor binding data and its applicability to the determination of active concentration

Protein concentration data are required for understanding protein interactions and are a prerequisite for the determination of affinity and kinetic properties. It is vital for the judgment of protein quality and for monitoring the effect of therapeutic agents. Protein concentration values are typically obtained by comparison to a standard and derived from a standard curve. The use of a protein standard is convenient, but may not give reliable results if samples and standards behave differently. In other cases, a standard preparation may not be available and has to be established and validated. Using surface plasmon resonance (SPR) biosensors, an alternative concentration method is possible. This method is called calibration-free concentration analysis (CFCA); it generates active concentration data directly and without the use of a standard. The active concentration of a protein is defined through its interaction with its binding partner. This concentration can differ from the total protein concentration if some protein fraction is incapable of binding. If a protein has several different binding sites, active concentration data can be established for each binding site using site-specific interaction partners. This review will focus on CFCA analysis. It will reiterate the theory of CFCA and describe how CFCA has been applied in different research segments. The major part of the review will, however, try to set expectations on CFCA and discuss how CFCA can be further developed for absolute and relative concentration measurements.     

How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags? A modelling study

The anisotropic component of the magnetic susceptibility tensor (Δχ tensor) associated with various paramagnetic metal ions can induce pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) in proteins, yielding valuable restraints in structural studies. In particular, PCSs have successfully been used to study ligands that bind to proteins tagged with a paramagnetic metal ion, which is of great interest in fragment-based drug design. To create easy-to-interpret PCSs, the metal ion must be attached to the protein in a rigid manner. Most of the existing methods for site-specific attachment of a metal tag, however, result in tethers with residual flexibility. Here we present model calculations to quantify the extent, to which mobility of the metal-binding tag can compromise the quality of the Δχ tensor that can be determined from the PCSs observed in the protein. Assuming that the protein can be approximated by a sphere and the tag is attached by a single tether, the results show that a single effective ?χ tensor can describe the PCSs and RDCs of the protein spins very well even in the presence of substantial tag mobility, implying that PCSs of ligands in binding pockets of the protein can be predicted with similar accuracy. In contrast, the quality of the PCS prediction for nuclear spins positioned above the surface of the protein is significantly poorer, with implications for studies of protein–protein complexes. The simulations probed the sensitivity of the effective Δχ tensor to different parameters, including length of the tether between protein and metal ion, protein size, type and amplitude of tag motion, tensor orientation relative to the protein and direction of tag motion. Tether length and amplitude of motion were identified as two key parameters. It is shown that the amplitude of tag motions cannot be quantified by simple comparisons of the effective Δχ tensor with the alignment tensor determined from RDCs.     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.