High recovery of prochymosin from inclusion bodies using controlled air oxidation

Refolding of proteins from inclusion bodies is a field of increasing interest for obtaining large amounts of active enzymes. Consequently, the development of inexpensive and scalable processes is required. This is particularly challenging in the case of eukaryotic proteins containing cysteines, which may form disulfide bonds in the native active protein. Previous studies have shown that the formation of disulfide bonds is essential for the refolding of prochymosin. In this work we demonstrate that air oxidation can be efficiently used for the refolding of prochymosin and that 48% of the unfolded protein can be recovered as active enzyme at a final protein concentration of 0.8 mg/ml. Refolding of the protein strictly correlates with the change in pH of the refolding solution. We were able to follow the degree of oxidative renaturation of the prochymosin by simply measuring pH. Thus, the scaling up of the refolding system under controlled conditions was easily achieved. Analyses of different substances as folding aids indicate that the use of L-arginine or neutral surfactants improves the recovery of active protein up to 67% of the initial protein. The overall results indicate that prochymosin can be efficiently and inexpensively refolded with high yields by controlled air oxidation.     

Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH

The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin.

Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.     

Denaturation studies on natural and recombinant bovine prochymosin (prorennin).

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.     

Both chaperone and isomerase functions of protein disulfide isomerase are essential for acceleration of the oxidative refolding and reactivation of dimeric alkaline protease inhibitor

Oxidative refolding of the dimeric alkaline protease inhibitor (API) from Streptomyces sp. NCIM 5127 has been investigated. We demonstrate here that both isomerase and chaperone functions of the protein folding catalyst, protein disulfide isomerase (PDI), are essential for efficient refolding of denatured-reduced API (dr-API). Although the role of PDI as an isomerase and a chaperone has been reported for a few monomeric proteins, its role as a foldase in refolding of oligomeric proteins has not been demonstrated hitherto. Spontaneous refolding and reactivation of dr-API in redox buffer resulted in 45% to 50% reactivation. At concentrations <0.25 microM, reactivation rates and yields of dr-API are accelerated by catalytic amounts of PDI through its isomerase activity, which promotes disulfide bond formation and rearrangement. dr-API is susceptible to aggregation at concentrations >25 microM, and a large molar excess of PDI is required to enhance reactivation yields. PDI functions as a chaperone by suppressing aggregation and maintains the partially unfolded monomers in a folding-competent state, thereby assisting dimerization. Simultaneously, isomerase function of PDI brings about regeneration of native disulfides. 5-Iodoacetamidofluorescein-labeled PDI devoid of isomerase activity failed to enhance the reactivation of dr-API despite its intact chaperone activity. Our results on the requirement of a stoichiometric excess of PDI and of presence of PDI in redox buffer right from the initiation of refolding corroborate that both the functions of PDI are essential for efficient reassociation, refolding, and reactivation of dr-API.     

Catalysis of creatine kinase refolding by protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.     

GroE facilitates refolding of citrate synthase by suppressing aggregation.

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.     

Functional implication of disulfide bond, Cys250 -Cys283, in bovine chymosin.

The reduction, carboxymethylation and mercuration of disulfide bond, Cys250-Cys283, located on the surface of bovine chymosin molecule resulted in the loss of about 25% of enzyme activity, suggesting that Cys250-Cys283 is not intimately involved in catalytic mechanism. Cys250 and Cys283 were substituted with Asp. and Ser. by site- directed mutagenesis of the structural gene coding for bovine prochymosin B. All three mutants (C250D/C283S, C250D, C283S) failed to be activated to chymosin in acid, indicating that Cys250-Cys283 might have some contribution to the correct refolding of the unfolded prochymosin.     

Chaperone activity of DsbC.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.     

Functional analysis of tunicamycin-inducible gene A polypeptide from Aspergillus niger.

Tunicamycin-inducible gene A polypeptide (TIGA) is a member of the protein disulfide isomerase (PDI) family and is suggested to facilitate the folding of nascent polypeptides. The functional properties of TIGA were investigated here. TIGA acted as an isomerase, catalyzing the refolding of denatured and reduced ribonuclease A. TIGA also exhibited chaperone activity in the refolding of denatured prochymosin but not in the refolding of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), indicating that it had substrate specificity with respect to chaperone activity. Detailed study with a series of thioredoxin-motif (trx-motif) mutants revealed that the 2 trx-motifs of TIGA were not equal in activity. The N-terminal trx-motif was more active than the C-terminal trx-motif, and the first cysteine in each trx-motif was necessary for isomerase activity.     

Functional properties of PDIA from Aspergillus niger in renaturation of proteins

Functional properties of protein disulfide isomerase A (PDIA) from Aspergillus niger were investigated using ribonuclease A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prochymosin as substrates. PDIA was shown to function as an isomerase catalyzing the refolding of denatured and reduced ribonuclease A. PDIA also exhibited trx-independent chaperone activity preventing the aggregation of reduced, denatured GAPDH, an enzyme lacking disulfide bonds. Both isomerase activity and chaperone function of PDIA were essential for the efficient refolding of the reduced, denatured prochymosin.     

Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes

Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.     

Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1alpha isoform are active in this reaction. Ero1 has a preference for the PDI-toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.     

Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.     

Chaperone characteristics of PDI-related protein A from Aspergillus niger

The functional properties of a novel protein, protein disulfide isomerase-related protein A (PRPA) from Aspergillus niger T21, have been characterized. (1) PRPA possesses disulfide isomerase activity. (2) In Hepes buffer, at substoichiometric concentrations, PRPA facilitates the formation of inactive lysozyme aggregates associated with PRPA (anti-chaperone activity); while at a high molar excess, PRPA inhibits aggregation by maintaining lysozyme in a soluble, yet inactive, state (chaperone-like activity). However, PRPA only exhibits chaperone-like activity during lysozyme refolding in phosphate buffer. (3) Experiments have indicated that disulfide cross-linkage is not required for the interaction between PRPA and lysozyme, and hydrophobic interaction may be responsible for PRPA effect on lysozyme. (4) Co-expression of PRPA and prochymosin in Escherichia coli leads to reduction of inclusion bodies, rendering part of prochymosin molecules soluble yet inactive. The structural and functional characteristics of PRPA suggest that PRPA may play an important role in protein folding, aggregation, and retention in the endoplasmic reticulum.     

Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5‐disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.     

Monoclonal antibody refolding and assembly: Protein disulfide isomerase reaction kinetics

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody (MAb) refolding and assembly which accompanies disulfide bond formation. The MAbin vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb intermediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant for a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.     

Function of disulflde bond Cys45-Cys50 of prochymosin

The conditions (temperature, time, pH) for solubilizing inclusion bodies of prochymosin mutant, Cys45Asp/Cys50Ser, are identical with those for the wild type. Moreover, they have similar oxidative refolding behavior. Under the same renaturation conditions both of them can undergo correct refolding leading to the formation of activable molecules. This is quite different from the mutant with deletion of Cys250-Cys283, indicating that Cys45-Cys50 contributes less to the correct refolding of prochymosin than Cys250- Cys283. However, deletion of Cys45-Cys50 results in a remarkable decrease of the thermostability of pseudochymosin, suggesting that this disulfide bond plays an important role in stabilizing enzyme conformation. The proteolytic (P) and milk-dotting (C) activities of the mutant of pseudochymosin, Cys45Asp/Cys50Ser, are lower than those of its wild counterpart. The C/P ratio of the former is onefold higher than that of the latter.     

Protein disulfide isomerase-mediated cell-free assembly of recombinant interleukin-12 p40 homodimers.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two subunits, p35 and p40. The disulfide-linked homodimer (p40)2 has been shown to be a potent IL-12 antagonist. In the present study, the p40 subunit was refolded from Escherichia coli inclusion bodies. Formation of (p40)2 was greatly increased in a redox buffer containing reduced and oxidized glutathione, but was not significantly affected by the cosolvents urea, GdnHCl or Chaps. While protein disulfide isomerase (PDI), GroEL/ES or DnaK/J/GrpE suppressed aggregation during refolding of p40, only DnaK/J/GrpE and PDI enhanced p40 dimerization. Oxidative assembly of p40 into (p40)2 by PDI, but not suppression of aggregation, was strongly dependent on inclusion of BSA in the refolding buffer. It is concluded that both chaperone-like and disulfide isomerase effects are essential for correct folding of p40 into dimers.