Optimal Refolding Research Articles

Increased yield of high purity recombinant human interferon-γ utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 μg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 × 10 7 to 4 × 10 7 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the β′ subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (V H) and light chain (V L) were used as the template for cDNA synthesis. The sequences were joined by the addition of a “linker” sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.

Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions

A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris (2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Angstroms structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen.

Isolation of aminoglycoside nucleotidyltransferase(2 ′′)-Ia from inclusion bodies as active, monomeric enzyme

Aminoglycoside nucleotidyltransferase(2 ′′)-Ia (ANT(2 ′′)) confers resistance to pathogenic bacteria against several aminoglycoside antibiotics including gentamicin, kanamycin, and tobramycin. The gene for this aminoglycoside-modifying enzyme has been cloned from a clinical isolate of Pseudomonas aeruginosa. This gene was inserted into an overexpression vector, the vector was then transformed into Escherichia coli BL21(DE3), and the protein has been isolated in the form of inclusion bodies. Optimal refolding conditions have been determined to be direct dilution of solubilized inclusion bodies into 0.1 M Tris–HCl, pH 8.5, 0.2 M KCl, 0.4 M l-arginine, and 5 mM reduced glutathione at 4 °C. The refolded enzyme is monomeric in solution and has similar kinetic properties and substrate selectivity to the enzyme isolated in soluble form.

Structure-based Analysis of GPCR Function: Conformational Adaptation of both Agonist and Receptor upon Leukotriene B4 Binding to Recombinant BLT1

We produced the human leukotriene B4 (LTB4) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% α-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB4 with Ka=7.8(±0.2)×108M−1 and a stoichiometric ratio of 0.98(±0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB4. We report evidence that both partners, LTB4 and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB4 conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.

Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2.

The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography. A systematic approach was then used to optimize refolding parameters. This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format. Refolding efficiency varied markedly. Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidized glutathione. After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E. coli, and crystals of the protease were grown. The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2.

The in vivo and in vitro Effects of Chicken Interferon a on Infectious Bursal Disease Virus and Newcastle Disease Virus Infection

The in vitro and in vivo effects of chicken interferon alpha on infectious bursal disease virus (IBDV) infection were investigated in this study. A cDNA of interferon alpha was first cloned from a Chinese strain chicken Shiqi by reverse transcription-polymerase chain reaction. The deduced amino acid sequence has one amino acid substitution with chicken interferon alpha 1 at residue 65 (N to S) and two amino acid substitutions with chicken interferon alpha 2 at residues 50 (N to S) and 58 (P to L), respectively. A prokaryotic expression system was employed to produce a large quantity of recombinant protein. Recombinant interferon was purified in a one-step process, and an optimal refolding process was devised. About 51% recombinant protein from inclusion bodies was refolded, and the final yield of the recombinant interferon reached 24.66 mg/liter culture. The recombinant interferon suppressed IBDV plaque formation in a dose-dependent manner and ameliorated IBDV and Newcastle disease virus infection in both specific-pathogen-free (SPF) and commercial chickens. The antiviral effect of interferon alpha is more significant in commercial chickens than in SPF chickens, and the route of administration affects the efficacy of interferon therapy. This is the first reported study of the effects of interferon alpha on IBDV infection.

29] Protein engineering by expressed protein ligation

By allowing the controlled assembly of synthetic peptides and recombinant polypeptides, expressed protein ligation permits unnatural amino acids, biochemical probes, and biophysical probes to be specifically incorporated into semisynthetic proteins. A powerful feature of the method is its modularity; once the reactive recombinant pieces are in hand and the optimal ligation conditions have been developed, it is possible to quickly generate an array of semisynthetic analogs by simply attaching different synthetic peptide cassettes--in most cases the synthetic peptides will be small and easy to make. From a practical perspective, the rate-determining step in the process is usually not the ligation step (it is based on a simple and efficient chemical reaction), but rather the generation of the reactive polypeptide building blocks. In particular, optimizing the yields of recombinant polypeptide building blocks can require some initial effort. However, it should be noted that the initial investment in time required to optimize the production of the recombinant fragment is offset by the ease and speed with which one can produce the material thereafter. In the example described in this chapter, the yield of soluble intein fusion protein was slightly better using the GyrA intein than for the VMA intein, although in both cases significant amounts of fusion protein were present in the cell pellet. Studies are currently underway to identify optimal refolding conditions for GyrA fusion proteins solubilized from inclusion bodies.

Effects of macromolecular crowding on protein folding and aggregation.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

Minimal and optimal mechanisms for GroE-mediated protein folding.

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES. Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced. Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate. The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate. The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL. Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and holo-chaperones.

Unfolding and refolding of active apple polyphenol oxidase

For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH 4) 2SO 4 and 100 μM CuSO 4. However, CuSO 4 had limited effect on the recovering of PPOr activity, whereas (NH 4) 2SO 4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases.

Hop Modulates hsp70/hsp90 Interactions in Protein Folding

Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90. We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase. We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate. Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system. Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90. Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1. In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90. Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70. Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23. Conversely, once p23 is bound to hsp90, Hop binding is diminished. These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.

Temperature Effects on Refolding and Aggregation of a Large Multimeric Protein Using Capillary Zone Electrophoresis

Capillary zone electrophoresis (CZE) equipped with laser-induced fluorescence detection (UFO) allows rapid and sensitive identification of folding and aggregation intermediates of P22 tailspike endorhamnosidase using the intrinsic tryptophan fluorescence. Conversion of the protrimer into the native tailspike protein, which is the rate-limiting step, can be quenched at 0 °C and results in a loss of the protrimer in the refolding pathway. Results obtained from CZE.UFD and polyacrylamide gel electrophoresis indicate the loss of folding intermediates through the adsorption of polypeptide chains onto the wall of the refoldingvial. Early aggregation intermediates are studied using a temperature-sensitive folding tailspike mutant which shifts productive folding to the aggregation pathway at restrictive temperatures. By monitoring refolding intermediates during the temperature shift experiments, the early monomeric intermediate is identified as the thermolabile junctional intermediate between the productive and aggregation pathways. After passing the stage for accumulation of thermolabile intermediate, an increase in refolding temperature from 10 to 35 °C is carried out for achieving the optimal refolding kinetics and yields of tailspike endorhamnosidase.

Investigation of refolding condition for Pseudomonas fluorescens lipase by response surface methodology

Response Surface Methodology (RSM) was used to investigate optimal refolding conditions of Pseudomonas fluorescens lipase which was expressed as inclusion body in E. coli. Three interacting factors, protein concentration, pH and guanidine hydrochloride (GdnHCl) concentration were selected as the variables of RSM. The protein concentration did not affect the refolding yield within the selected range (50–340 μg ml −1) at low temperatures of 4 and −15°C, but it was a critical factor at 25°C and refolding yield significantly decreased with increasing protein concentration. The pH and GdnHCl were significant factors in all experimental conditions. But there was no trends of optimal pH depending on temperature and additives, just showing the optimum at neutral pH. Glycerol shifted optimal GdnHCl concentration to higher side, while arginine to lower side. Therefore, it was concluded that glycerol and arginine deprives and supplements GdnHCl, respectively. In this study, RSM made it possible to investigate successfully the optimal conditions of in vitro refolding and to elucidate interactions between refolding factors with a minimum number of experiments. Under the optimal condition determined by RSM, approximately 90% refolding yield was obtained and it was a 30% increase over the conventional method.

Relationship between the optimal temperature for oxidative refolding and the thermal stability of refolded state of hen lysozyme three-disulfide derivatives.

The temperature dependence of the efficiency of oxidative refolding was examined for hen lysozyme three-disulfide derivatives produced in Escherichia coli. Each derivative was designed to lack one of the four disulfide bridges in authentic lysozyme: delta 1 (Cys6-->Ser, Cys127-->Ser), delta 2 (Cys30-->Ser, Cys115-->Ser), delta 3 (Cys64-->Ser, Cys80-->Ser), delta 4 (Cys76-->Ser, Cys94-->Ser), delta 2Ala (Cys30-->Ala, Cys115-->Ala), and delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for delta 1 (19 degrees C) and highest for delta 4Ala (30 degrees C). The chromatographically purified, completely refolded three-disulfide species were not stable above the optimal refolding temperature in the presence of glutathione. The stability of each of them was determined from the far-UV CD thermal denaturation measurement at pH 3.9 in the absence of glutathione, where the denaturation was reversible. The transition temperature was lowest for delta 1 and highest for delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were found to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for delta 1 in detail. The optimal temperature for refolding increased by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability in the presence of glycerol of refolded and purified delta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese

In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins. The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese. These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671). The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60. The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10. Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP. At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins. This spontaneous refolding can be arrested by chaperonin 60. There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP. ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins. Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). The chaperonin proteins appear to play a similar role in as much as they can replace the detergents. Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60. The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added. The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.