A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.     

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.     

Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration

The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.     

Cooperative effect of artificial chaperones and guanidinium chloride on lysozyme renaturation at high concentrations

It has been recognized that the artificial chaperone system, cetyltrimethylammonium bromide and beta-cyclodextrin, is effective for enhancing protein renaturation. In this work, we studied the effect of the artificial chaperone system and guanidinium chloride (GdmCl) on the oxidative renaturation of lysozyme at 0.21-1.05 mg/mL, and a kinetic model based on the competition between protein folding and aggregation was employed to express the renaturation process. The refolding rate constant increased, while the aggregation rate constant decreased, with increasing concentration of the artificial chaperones. With increasing GdmCl concentration (0.28-2 M), both rate constants decreased, but there existed a specific GdmCl concentration that maximized the ratio of the two rate constants and thus the renaturation yield. The results obviously indicated the cooperative effect of GdmCl and the artificial chaperones on enhancing protein renaturation.     

Folding of the firefly luciferase polypeptide chain with immobilized C-terminus

Refolding of Photinus pyralis firefly luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of the enzyme is fast and productive in the absence of chaperones [Svetlov et al., 2006. Protein Sci. 15, 242-247]. During cotranslational folding, the C-termini of polypeptides are bound to massive particles - ribosomes. The question arises whether the immobilization of the polypeptide C-terminus on a massive particle promotes the folding. To test this experimentally, the luciferase with oligohistidine tag at its C-terminus was prepared. This allowed us to immobilize the protein C-terminal segment on chelating Sepharose beads. Here we show that both immobilized and free chains of urea-denatured enzyme refold with the same rate. At the same time, the immobilization of luciferase results in higher refolding yield due to prevention of inter-molecular aggregation. Chaperones of the Hsp70 family promote refolding of both immobilized and free luciferase polypeptides. The results presented here suggest that the high rate of cotranslational folding is not caused by the immobilization of polypeptide C-termini by itself, but is rather due to a favorable start conformation of the growing polypeptide in the peptidyl-transferase center of the ribosome and/or the strongly vectorial character of the folding from N- to C-terminus during polypeptide synthesis.     

The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient

Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein-protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.     

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.     

Production and purification of firefly luciferase inEscherichia coli

Summary A genetic recombinant stain ofE.coli were induced to express and secrete firefly luciferase. Cells, when broken by freeze/thawing, gave about 2% of the total soluble protein as luciferase. The luciferase was purified with ammonium sulphate fractionation and gel filtration chromatography giving a luciferase product with high specific activity (10⁶ light units/mg protein). SDS-PAGE of this product showed two active bands at 54 and 50 kDa, which corresponded to the luciferases with and without a signal peptide on their N-terminals. The yield was more than one mg purified enzyme per 100 ml of fermentative liquid.     

Folding of firefly luciferase during translation in a cell-free system.

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell-free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell-free system while this protein is being translated from its mRNA. It is shown that ribosome-bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half-time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.     

Folding of the firefly luciferase polypeptide chain with the immobilized C terminus

Refolding of firefly Photinus pyralis luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of luciferase is fast and productive in the absence of chaperones. During cotranslational folding, the C termini of polypeptides are associated with ribosomes, massive particles. The question arises as to whether C-terminal immobilization on a massive particle promotes folding. To study this problem experimentally, luciferase was C-tagged with hexahistidine to allow its C-terminal immobilization of chelating Sepharose. Both immobilized and free chains of the urea-denatured enzyme refolded at the same rate. At the same time, immobilization led to a higher refolding yield owing to the prevention of intermolecular aggregation. Chaperones of the Hsp70 family promoted folding of both immobilized and free luciferase polypeptides. It was assumed that the high rate of cotranslational folding is not ensured by mere immobilization of the C terminus of the polypeptide, but is rather due to a favorable start conformation of the growing peptide in the peptidyltransferase center of the ribosome and/or the vectorial character of the folding, proceeding from the N to the C end during polypeptide synthesis.     

Optimization of luciferase activity in a tomato transient assay system

The gene encoding firefly luciferase is a commonly used reporter for transient expression assays in plants. We have found that the concentration of buffers normally used in luciferase assays is too low to adequately buffer acidic plant organs. This results in low apparent luciferase activity as well as high variability among replicates. In a transient assay system based on particle bombardment of ripe tomato fruit, luciferase activity driven by the 35S promoter of cauliflower mosaic virus was increased as much as 130 fold by increasing the concentration of the buffer from 50 mM to 300 mM. Using 300 mM buffer, expression levels of luciferase driven by three different plant promoters were found to reflect expression patterns in intact plants.     

High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase

Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor=0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microg/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <10 microM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.     

Oxidative renaturation of lysozyme at high concentrations

Newly synthesized cloned gene proteins expressed in bacteria frequently accumulate in insoluble aggregates or inclusion bodies. Active protein can be recovered by solubilization of inclusion bodies followed by renaturation of the solubilized (unfolded) protein. The recovery of active protein is highly dependent on the renaturation conditions chosen. The renaturation process is generally conducted at low protein concentrations (0.01-0.2 mg/mL) to avoid aggregation. We have investigated the potential of successfully refolding reduced and denatured hen egg white lysozyme at high concentrations (1 and 5 mg/mL). By varying the composition of the renaturation media, optimum conditions which kinetically favor proper folding over inactivation were found. Solubilizing agents such as guanidinium chloride (GdmCl) and folding aids such as L-arginine present in low concentrations during refolding effectively enhanced renaturation yields by suppressing aggregation resulting in reactivation yields as high as 95%. Quantitatively the kinetic competition between lysozyme folding and aggregation can be described using first-order kinetics for the renaturation reaction and third-order kinetics for the overall aggregation pathway. The rate constants for both reactions have been found to be strongly dependent on denaturant and thiol concentration. This strategy supercedes the necessity to reactivate proteins at low concentrations using large renaturation volumes. The marked increase in volumetric productivity makes this a viable option for recovering biologically active protein efficiently and in high yield in vitro from proteins produced as inclusion bodies within microbial cells. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 221-230, 1997.     

生物素化荧光素酶的克隆表达及其固定化研究

为了在体内实现萤火虫荧光素酶的生物素酰化修饰,我们将大肠杆菌中编码生物素羧基载体蛋白（biotin carboxyl carrier protein,BCCP）C端87个氨基酸的功能域基因融合到萤火虫(Pyrocoelia pectoralis)荧光素酶cDNA的末端。经大肠杆菌生物素合成酶（biotin holoenzyme synthetase）的催化,生物素与BCCP上特定的赖氨酸（Lys）残基共价结合,由此与BCCP融合的萤火虫荧光素酶间接实现了生物素化的修饰。利用生物素与配体亲和素或链霉亲和素的特异性耦合,可以将荧光素酶固定到亲和素或链霉亲和素包被的磁珠上,从而使荧光检测的应用更加灵活和方便。本文将就生物素化荧光素酶的克隆、表达以及功能检测进行具体讨论。     

On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment

The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Physical-chemical features of non-detergent sulfobetaines active as protein-folding helpers

Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and beta-D-galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Suppresses Aggregation of Recombinant Growth Hormones in Refolding Process from <Emphasis Type="Italic">E. coli</Emphasis> Inclusion Bodies

l-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from E. coli inclusion bodies by solubilization–dilution protocol at high protein concentration and pH 8.0. The influence of l-arginine concentration on the renaturation yield of both proteins was investigated. l-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented.     

Potent lectin-independent chaperone function of calnexin under conditions prevalent within the lumen of the endoplasmic reticulum

Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site with specificity for Glc1Man9GlcNAc2 oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated substrates at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. Here, we examine the ability of calnexin to interact with a non-glycosylated substrate under physiological conditions of the ER lumen. We show that the soluble ER luminal domain of calnexin can indeed suppress the aggregation of non-glycosylated firefly luciferase at 37 degrees C and at the normal resting ER calcium concentration of 0.4 mM. However, gradual reduction of calcium below the resting level was accompanied by a progressive loss of native calnexin structure as assessed by thermal stability, protease sensitivity, intrinsic fluorescence, and bis-ANS binding. These assays permitted the characterization of a single calcium binding site on calnexin with a Kd = 0.15 +/- 0.05 mM. We also show that the suppression of firefly luciferase aggregation by calnexin is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to calnexin in the presence of 0.4 mM calcium is 0.7 mM. ATP did not alter the overall stability of calnexin but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings demonstrate that calnexin is a potent molecular chaperone that is capable of suppressing the aggregation of substrates through polypeptide-based interactions under conditions that exist within the ER lumen.     

Expression and purification of polyhistidine-tagged firefly luciferase in insect cells--a potential alternative for process scale-up

The coleopteran firefly, Photinus pyralis, luciferase was produced in lepidopteran Trichoplusia ni insect cells using a baculovirus expression vector. The recombinant protein was equipped with a polyhistidine affinity tag at the carboxyl terminus and purified by immobilized metal-ion affinity chromatography in combination with an expanded bed adsorption system. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. According to light emission measurements, the final yield of highly purified protein was 23 mg l(-1) of cell culture. In addition, no specific interaction of interfering substances, such as, ATP, adenylate kinase, nucleoside diphosphokinase, as well as, creatine kinase of the final preparation were identified. Together, the results presented here clearly show that the baculovirus expression system in combination with immobilized metal-ion affinity chromatography is a potential strategy for process scale-up of polyhistidine tagged insect luciferase.