Renaturation Process Research Articles

Synthesis, purification and biological properties of a truncated mutant form of human tissue plasminogen activator produced in E. Coli

A truncated form of tissue plasminogen activator (t-PA) that lacks the two N-terminal domains of wild type t-PA was efficiently renatured and purified after expression in E. coli. The mutant t-PA was more soluble than wild type t-PA expressed as a nonglycosylated form in E. coli as was predicted by the elimination of a significant segment of predominantly hydrophobic sequence in the mutant. The efficiency of. the renaturation process was, however, unchanged. In vitro biological activity of wild type and mutant t-PA was similar in chromogenic, fibrin plate and clot lysis assays. Both proteins also produced specific plasminogen activation in rabbits, however clearance of the mutant from the circulation was about four times slower than that of wild type t-PA. This confirms previous observations that the N-terminal domains contribute to the rapid clearance of t-PA, but are not essential for specific plasminogen activation in vivo.

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a M r of 40,000. The M r of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by l-methionine and l-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 m guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.

Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.

Structural and functional studies of the rat mitochondrial single strand DNA binding protein P16

The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found. Measurement of the affinity of P16 for isolated sites by titration with either oligo(dT) 8 or 5′-dephosphorylated oligo(dT) 8 indicated values on the order of 10 7 m −1. P16 exhibited a binding preference for single strand DNA, poly(dT), and poly(dC) in comparison to double strand DNA, poly(U), or poly[d(A-T)]. Although it was not possible to show that P16 destabilizes double helical DNA or even poly[d(A-T)], binding of P16 does inhibit the process of renaturation as shown by inhibition of duplex formation between poly(dA) and poly(dT). The binding of saturating amounts of P16 to single strand poly(dT) · oligo(dA) 50 template-primers enhanced approximately 10-fold the activity of both the homologous mitochondrial DNA polymerase and the Escherichia coli DNA polymerase I Klenow fragment. However, the mitochondrial DNA primase was nearly completely inhibited by the saturation of the poly(dT) template with P16. Amino-terminal sequence analysis of P16 and a protease-insensitive, DNA binding domain ( M r ≈ 6000) revealed that the DNA binding domain resides, at least in part, in the amino-terminal third of the P16 molecule. Furthermore, the amino-terminal sequence was found to be strikingly similar to that of the Xenopus laevis mtSSB-1 and to a lesser extent similar to E. coli SSB and E. coli F sex factor SSB.

The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co 2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co 2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The binding mechanisms of Co 2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.

Denaturation by urea and renaturation of 20β-hydroxysteroid dehydrogenase studied by high-performance size exclusion chromatography

The denaturation by urea and renaturation of 20β-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH high molecular weight aggregates, native tetramers and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.

A physical-chemical and immunological comparison shows that native and renatured Escherichia coli tryptophan synthase ß2 subunits are identical

An acid-denaturation of the ß 2 subunit of Escherichia coli tryptophan synthase has been recently described. In the present study, renaturation yield of acid-denaturated ß 2, and the influence of temperature, protein concentration and presence of ligands are investigated. It is also demonstrated that 3 forms of the protein are obtained at the end of the renaturation process: one is fully active, and is identical to native ß 2, as indicated by some of its chemical and physical properties, as well as by its immunological reactivity towards monoclonal antibodies specific for the native protein. A second form is composed of high molecular weight insoluble and inactive aggregates. A third form consists of low molecular weight soluble and inactive aggregates. The results obtained for the immunochemical reactivity of these small aggregates indicate that they are formed with partly correctly folded β monomers assembled by specific but incorrect quaternary interactions. The capicity of monoclonal antibodies to detect such incorrect structures and to characterize renatured proteins is particularly emphasized.

Polarographic studies on renaturation of urea-denatured human serum albumin

The renaturation of the two main components of human serum albumin, i.e. of mercaptalbumin and nonmercaptalbumin, was studied polarographically. It has been demonstrated that renaturation of both proteins after 1-min denaturation in 8M urea is reversible. By contrast, renaturation after 200 min denaturation in 8M urea is an irreversible process; the characteristics of renatured mercaptalbumin differ more from the properties of the native protein than the characteristics of nonmercaptalbumin. The studies of the kinetics of renaturation of both proteins have shown that the renaturation can be represented by a two-state model. This means that the existence of stable intermediary products during the renaturation process was not determined polarographically.

Mass spectrometric analysis of recombinant human α-2 interferon

Mass spectrometry (MS) was used for the characterization of recombinant human α-2 interferon (α-2 IFN) produced in E. coli. After purification by monoclonal antibody affinity chromatography, α-2 IFN showed two major peaks in reversed-phase liquid chromatography (RP- LC). Each component was digested with trypsin and Staphylococcus aureus protease V8, separately or in tandem, and the peptide mixture was analysed by MS without further purification. The first peak corresponded to the 165 amino acid sequence of human α-2 IFN and the main component of the second peak was the acetylated Cys 1 α-2 IFN. It was also possible to verify by MS the location of the SS bonds in α-2 IFN and the occurrence of incorrect SS bridges in the products of some renaturation processes. The best renaturation process for obtaining a product without adducts or scrambling of disulphide bonds could be found by using RP-LC and fast atom bombardment MS.

Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its alpha and beta subunits.

Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an alpha 2 beta 2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding alpha subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer compositions that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.

Investigation of fenaturation in gelatin gels

This paper contributes to the description of the different phases involved in the gelation of aqueous gelatin solutions whichis managed by the coil-helix transition of gelatin chains. The viscosimetric beahviour versus ageing time shows an induction period during which viscosity has immeasurable value. This induction period appears to be strongly dependent on concentration and quench temperature. The end of induction has been found to occur at a common helix concentration independent of quench temperature and initial concentration. The analysis of these results by classical kinetics agrees with a cooperative mechanism involving the formation of intermolecular triple helices. The first stage of helix content evolution deduced from polarimetric or ultrasonic absorption measurements has been analysed by the Avrami theory. The primary stage of the renaturation process can be considered as a conventional crystallization process consisting in a rapid nucleation followed by an unidirectional growth.

Subunit interactions of 7 S nerve growth factor. Gamma-esterase activity, rates, and conformational changes during reassociation.

The gamma-esterase activity of 7 S nerve growth factor (NGF) is depressed relative to free subunit because of constraints within the oligomer. 4 M urea causes a reversible dissociation of 7 S NGF and concomitant increase in esterase activity. The gamma-esterase activity with tosyl-arginyl-methyl-ester increased between 1 and 4 M urea and was inhibited at higher concentrations. Upon dilution of 7 S NGF from 6 M urea, the esterase activity was initially identical with that of the similarly treated gamma-subunit but decayed rapidly (10 min) to approach that of the native 7 S NGF. In the presence of 100 microM EDTA the activity was higher, but still required the same length of time to reach a constant depressed value. Upon dilution of 7 S NGF from 2 M NaCl the rapid decay was not observed and the activity remained constant at about the same level as the equilibrium values for the urea-treated and renatured 7 S NGF. A red shift in the fluorescence maximum and a concomitant increase in quantum yield occurred for the 7 S in 8 M urea. Dilution into buffer resulted in a rapid decay and return to native 7 S fluorescence but not when EDTA was included. Both the decay in esterase activity and in fluorescence intensity upon dilution from urea were first order reactions with a rate constant of about 8 X 10(-3) s-1 suggesting that both methods were measuring a unimolecular renaturation process. Renaturation from NaCl or recombination of isolated subunits was much more rapid, indicating a simple combination of subunits in a native conformation. The circular dichroism spectra of urea and NaCl-treated 7 S NGF were different, and spectra of the renatured species with and without EDTA differed. Reassociation to a conformationally different 7 S NGF probably occurs in the presence of EDTA. The results emphasize the role of zinc in 7 S NGF formation and the influence of conformational changes in the effect of beta-NGF on the activity of the gamma-esterase in 7 S NGF.

Stability of VIII:C in plasma: The dependence on protease activity and calcium

Loss of Factor VIII procoagulant activity (VIII:C) following blood collection is a major problem in providing sufficient amounts for therapeutic use and biochemical analyses. We have examined the effects of inhibition of plasma proteases and maintenance of physiological calcium ion on plasma VIII:C stability. The addition of protease inhibitors such as benzamidine, phenylmethylsulfonyl fluoride (PMSF), aprotinin, or soybean trypsin inhibitor (SBTI) to CPD plasma provided no significant protection against decay of VIII:C activity. Neither the rate of decay in the first 24 hours nor the final VIII:C activity observed after storage for 48–72 hours were significantly altered. On the other hand, addition of DFP or heparin to CPD plasma resulted in a marked improvement in VIII:C stability over 24 hours. This demonstrated that these two inhibitors are effective in preventing VIII:C degradation during storage. In addition to protease inhibition, the importance of maintaining physiological calcium ion was demonstrated by 100% stabilization of VIII:C in heparin plasma. Plasma obtained from CPD plus heparin blood could also be stabilized provided free calcium ion levels were restored to physiological concentrations. The inactivation of VIII:C in CPD plus heparin plasma was completely reversible up to 4 hours after collection. Studies on the recovery of activity after recalcification of CPD plus heparin plasma provided kinetic data which support a renaturation process of VIII:C rather than one due to enzymatic activation. The use of a thrombin-specific chromogenic substrate revealed that after recalcification and during the recovery of VIII:C activity, there was no significant thrombin activity. Although the data suggest that proteolytic degradation plays some part in VIII:C decay, only the maintenance of physiologic calcium ion levels under cover of an effective non-chelating anticoagulant and protease inhibitor allows preservation of VIII:C activity.

Investigation into the ageing process in gels of gelatin/water systems by the measurement of their dynamic moduli

During the ageing (renaturation) process of 1–5% aqueous gelatin solutions the dynamic moduli were measured in the temperature range from −1.2 °C to 25.2 °C. The renaturation process appears to be strongly dependent on concentration, temperature and thermal history. The process at 25.2 °C appears to be of 3rd order, whereas the rate determining step appears to be the aggregation of mono helices into triple helices.

L-Phenylalanine stereospecifically inhibits the renaturation of muscle pyruvate kinase

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine HCl. Subsequent dilution of the enzyme into buffer containing β-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with an average half-time of 17 min at 16 °C. The addition of 1 m m l-phenylalanine increases the average half-time for recovery of enzymatic activity to 26 min, while 8 m m l-phenylalanine further increases this value to 46 min. Tyrosine and tryptophan also inhibit the reactivation but to a lesser extent than phenylalanine. Neither l-alanine, l-valine, d-phenylalanine, phosphoenolpyruvate, nor fructose 1,6-bisphosphate have any appreciable effect on activity recovery rates, either in the presence or absence of l-phenylalanine. Phenylpyruvate is a very potent inhibitor of reactivation. The addition of 5 m m phenylpyruvate increases the half-time to 57 min. The evidence presented in this paper supports the hypothesis that an l-phenylalanine-binding site which probably is distinct from the catalytic site is formed early in the renaturation process. l-Phenylalanine binds to this site and inhibits two first-order relaxations that are rate limiting for the reactivation and that have the following rate constants: 8.76 × 10 −2 and 1.24 × 10 −2 min −1, respectively, in the absence of phenylalanine and 3.04 × 10 −2 and 7.63 × 10 −3min −1, respectively, in the presence of 8.0 m m phenylalanine. We presume these first-order processes to be transconformational steps in the reactivation process.

Refolding of a three (noncovalently linked)-domain enzyme. Human gamma-thrombin.

Human gamma-thrombin is a three (noncovalently linked)-domain enzyme which contains the known serine protease catalytic triad, Asp-His-Ser, one on each of the three noncovalent domains (Asp 99 on the A-B3 chain). While protein-folding dogma does not necessarily predict that the denatured form of this enzyme could refold to the correct conformation, a monitor of the esterase activity (Tos-Arg-OMe) shows complete recovery of native catalytically active conformation. When compared with the covalently intact alpha form which refolds from urea in less than 2 min with complete return of both clotting and esterase activity, gamma-thrombin requires up to 90 min to regain full esterase activity. The gamma-thrombin reactivation data best fit a single first order rate constant, k = 0.03 +/- 0.005 min-1. It was suggested that the gamma-thrombin renaturation process might represent first the rapid refolding, then subsequent reassociation and reisomerization of the three noncovalent domains to yield a lower energy, fully active, conformation. This study represents the only example known of the refolding (reconstitution) of a three (noncovalent)-domain protein.

Studies of the structure-function relationships of Neurospora pyruvate kinase: Denaturation by urea and the effect of ligands on the refolding and renaturation process

1. 1. The renaturation of Neurospora pyruvate kinase following denaturation with urea was investigated. 2. 2. The substrates, phosphoenol pyruvate and adenosine diphosphate, were observed to stabilize the enzyme against urea-induced structural disorganization. 3. 3. It was demonstrated that for refolding/reactivation of the denatured protein the allosteric activator, fructose-1,6-diphosphate and a sulphydryl protectant are required. 4. 4. The enzyme recovered following renaturation showed complete immunological identity with the native enzyme in Ouchterlony double diffusion tests.

Refolding and Reactivation of Liver Alcohol Dehydrogenase after Dissociation and Denaturation in 6M Guanidine Hydrochloride

Horse-liver alcohol dehydrogenase has been dissociated and denatured by 6 M guanidinium hydrochloride. Removal of the denaturant under optimum conditions of the solvent leads to partial reactivation. The concentrations of the enzyme, as well as the coenzyme (NAD+), and Zn2+, affect the reactivation significantly, since high concentrations promote the formation of inactive aggregation products. Analyzing the kinetics of reactivation and reassociation, conditions far from equilibrium of dissociation-association provide maximum yields (approximately 70%). The sigmoidal kinetic traces suggest a superposition of first-order transconformation and second-order association reactions; the latter are corroborated by the concentration dependence of the reactivation reaction. The coenzyme, NAD+, has no influence on the kinetics of reactivation. Addition of Zn2+ leads to a significant decrease of the rate and yield of reactivation. The process of renaturation, as reflected by the regain of native fluorescence shows complex kinetics: rapid relaxations are followed by slower first-order and second-order processes which parallel reactivation.

Methods of denaturation and renaturation of DNA in interphasic chromatin: cytochemical quantitative analysis by Methyl Green staining.

Almost diploid nuclei (as judged from the microdensitometric evaluation of the Feulgen positive material) of granular and Purkinje cells of the rat cerebellar cortex, were submitted to in situ DNA denaturation and renaturation experiments. We assessed the double-strandedness of DNA, by Methyl Green staining according to Scott (1967). Under these conditions a stoichiometric ratio between bound dye and DNA exists, suitable for quantitative microdensitometric measurements. Our data show that DNA in the interphasic chromatin is never completely denatured after the treatments we used. Furthermore, the renaturation takes place in a different way in the two cell types. Owing to the unlike chromatin packing of granular and Purkinje nuclei, we suggest that nuclear proteins must interfere differently on the in situ denaturation and renaturation processes.

Kinetics of the renaturation of yeast tRNA3Leu

AbstractYeast tRNA3Leu is one of several tRNA molecules which can adopt a stable, biologically inactive, denatured conformation. The circular dichroism of the native and denatured conformers differs, providing the basis for the present study of the mechanism for the renaturation process. Conversion of the denatured structure to the native takes place in two steps: a rapid change occurring immediately on addition of Mg++, followed by a slower, strongly temperature‐dependent step which returns the molecule to its biologically active state. Optimal kinetic data for the second step could be obtained at 285 nm. Analysis of the time dependence of Δε285 by the Guggenheim method demonstrated that this step follows first‐order kinetics. The temperature dependence of the rate constants over the range 32–41°C yielded the following parameters for the rate‐limiting step: Ea = 69 kcal/mole, ΔH‡ = 69 kcal/mole, and ΔS‡ = 146 cal/mole deg. Values of this magnitude are typical of order—order transitions in nucleic acids.