Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease

Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis. Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen IV, laminin, and fibronectin. A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity that could be very helpful in thrombosis therapy. We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library. The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at either 37 or 20 degrees C led to the overexpression of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction. The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharose column followed by gel filtration chromatography, both under denaturing conditions. After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The refolded recombinant protein was active in fibrin-agarose plates. The purified protein achieved a conformation similar to that of the native enzyme as judged by circular dichroism analysis.     

Human and mouse macrophage-inducible C-type lectin (Mincle) bind Candida albicans

Candida albicans is a causative agent in mycoses of the skin, oral cavity, and gastrointestinal tract. Identification of receptors, and their respective ligands, that are engaged by immune cells when in contact with C. albicans is crucial for understanding inflammatory responses leading to invasive candidiasis. Mincle is a recently identified macrophage-expressed receptor that is important for host responses to C. albicans. The carbohydrate-recognition domain of human and mouse Mincle were expressed, purified under denaturing conditions, and successfully refolded. In addition to oligomers, there are isolatable monomeric and dimeric forms of the protein that occur under two different buffer solutions. The human and mouse homologues bound yeast extract, and the isolated dimeric and monomeric species also demonstrated the recognition of whole C. albicans yeast cells. The data are indicative of several functional states mediating the interaction of Mincle and yeast at the surface of the macrophage.     

Production and recovery of recombinant propapain with high yield

Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a⁺ each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400 mg of properly refolded propapain was obtained from 1 L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.     

Histone H1: Ultracentrifugation studies of the effects of ionic strength and denaturants on the solution conformation

The conformation of histone H1 has been examined under native and denaturing conditions in the absence of DNA or chromatin. Sedimentation coefficients were determined for Histone H1 in 0.1 m KCl and in 6 m guanidine hydrochloride solutions at pH 7.4. The influence of ionic strength on the conformation of histone H1 has been determined by measurement of the sedimentation coefficient in tetramethylammonium chloride solutions of up to 2.5 m and extrapolated to infinite ionic strength. Results from these experiments suggest that the native conformation of histone H1 is very asymmetric in shape. The molecule is best described as a prolate ellipsoid with axes of 312 Å (2a) and 16 Å (2b) in low ionic strength media and also as a prolate ellipsoid with axes of 202 Å (2a) and 20 Å (2b) at high ionic strength or when associated with polyanions, e.g., DNA. Denaturation of histone H1 by guanidine hydrochloride was found to be completely reversible. In 6 m guanidine hydrochloride, the H1 molecule collapses to a sphere but the original extended conformation of the protein is readily restored on dialysis. This suggests rigid conformational requirements for the H1 molecule as incorporated into chromatin. The shape and dimensions for the H1 molecule at high ionic strength are not sufficiently conclusive to locate H1 in the chromatin structure. It is proposed, however, that viable models for chromatin architecture must be consistent with the histone H1 solution dimensions obtained here.     

Structure of human tumor necrosis factor alpha derived from recombinant DNA

Recombinant DNA derived tumor necrosis factor alpha, when expressed at a high level in Escherichia coli, appeared in the pellet and soluble fractions of disrupted cells. The protein was purified from the pellet fraction by solubilizing it in urea and reducing agent and was refolded into a buffer without these additives. The structure of the protein was identical with that purified from the soluble fraction without exposure to both reducing and denaturing agents, as demonstrated by circular dichroism, gel filtration, and sulfhydryl titration. As a reflection of the structural similarity, both purified proteins showed identical cytolytic activity on mouse L929 cells. The protein was characterized as an essentially nonhelical and beta-sheet-rich structure and possibly as a noncovalently associating oligomer. Two cysteine residues form an intrapolypeptide disulfide bond.     

Calcium,Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.     

Immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections

Three different variants of the recombinant hybrid outer membrane protein OprF (aa 190-342)-OprI (aa 21-83) could be obtained in high yield after expression in Escherichia coli. The hybrid protein was modified N terminally, either with a minimal histidine tag or with a homologous sequence of OprF. Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions, and this produced three suitable candidates for a vaccination trial, protein His-F-I, which was purified in its native as well as in its refolded form; and the native purified N terminally extended protein, ex-F-I. In mice, significantly higher antibody titers and survival rates after challenge with Pseudomonas aeruginosa were observed following immunization with protein His-F-I, purified under native conditions.     

Intensity fluctuation spectroscopy and transfer RNA conformation. III. Influence of NaCl concentration on the size and shape of the initially salt-free tRNA in solution.

The influence of sodium ion concentration in solution on the initially salt-free conformation of bulk tRNA from baker's yeast has been investigated by means of photon correlation spectroscopy. From the measured values of translational (D^T) and rotational (D^R) diffusion coefficients, the semiaxes of an ellipsoid of revolution, which are hydrodynamically equivalent to the tRNA molecule, were calculated for tRNA solutions in pure H₂O as well as in 0.005, 0.1, 0.5M NaCl and 0.01M MgCl₂ solutions at pH 4.2 and 7.5. These data, combined with our previous studies, suggested a model which describes the formation of an ordered tRNA structure due to increasing NaCl concentrations. Furthermore, we have obtained information concerning intermolecular interactions between tRNA molecules in solution. In low-salt or salt-free tRNA solutions, we detected in the linewidth distribution function an extra-fast component which can be attributed as possibly due to charge fluctuations related to the reaction of ionization of organic bases. In our light-scattering linewidth measurements, we do not see fluctuations of charged and uncharged states directly as concentration fluctuations. Rather, we postulate a modulation of long-range intermolecular electrostatic interactions between the tRNA molecules due to such charge fluctuations. It is this modulation which is related to the fast component of the time correlation function at finite concentrations. A quantitative theory is needed to provide a more definitive explanation of the dynamical behavior of tRNA in salt-free or low-salt solutions.     

Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case

It has recently been reported that 2‐methyl‐2,4‐pentanediol (MPD) can modulate the protein‐binding properties of sodium dodecyl sulfate (SDS), turning it into a non‐denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD‐based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far‐UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20–40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417–423. © 2012 Wiley Periodicals, Inc.     

Site-specific pegylation of G-CSF by reversible denaturation

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.     

Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut

In this work, we describe the expression, purification, and disulfide mapping of the named 'peanut seed cDNA 33' (PSC33) peanut allergen. A variant of PSC33 (with N(63), E(64), Q(69) instead of D(63), Q(64), E(69)) has been identified in peanut by proteomic analysis of a highly IgE immunoreactive purification fraction. It is 92% homologous to Ara h 6. We raised monoclonal antibodies against PSC33 and amplified it by PCR from peanut leaf genomic DNA. PSC33 was intron-less and the two NEQ and DQE variants of PSC33 were equally amplified. Since expression of the natural PSC33 (DQE) gene was very low in Escherichia coli even with supplementation of rare codon tRNAs, a synthetic gene optimized for expression in E. coli of PSC33 (DQE) was introduced into a pET9-c vector. A high production of protein occurred in the inclusion bodies that was submitted to refolding using an additive-introduced stepwise dialysis protocol which consists in the gradual removal of the denaturing agent guanidine-HCl with controlled introduction of oxidized and reduced glutathione and l-arginine as a chemical chaperone. After reverse phase HPLC purification, 1mg of pure refolded protein (as assayed by MALDI-TOF mass spectrometry, mouse IgG immunoreactivity and circular dichroism) were obtained with every 100ml of bacterial culture. Trypsin and CNBr hydrolysis of the protein combined with MALDI-TOF mass spectrometry allowed us to assign disulfide bridges and show that the native and refolded proteins were identical. The four disulfides of canonical 2S albumins were conserved and the two supplementary cysteines of PSC33 were paired together.     

Structural restoration of inactive recombinant fish growth hormones by chemical chaperonin and solvent restraint approaches

Recombinant proteins may undergo conformational distortion, leading to aggregation and loss of function, when they are expressed in heterologous systems. The structural and functional restoration of such inactive proteins is highly desirable. We have over-expressed recombinant growth hormones from the fish ayu (Plecoglossus altivelis) and yellow grouper (Epinephelus awoara) by a pET expression system. Both recombinant proteins accumulate as insoluble form in Escherichia coli. We refolded these inactive proteins into the active form using a stepwise refolding process with a dilute denaturing agent as a steric blocker and chemical chaperonin. Optical characterization showed that stable folding intermediates with a helical conformation can be detected in the molten globule state. Moreover, the function of restored recombinant growth hormones was demonstrated by its ability to stimulate proliferation in zebrafish liver cells.     

Optimized expression and refolding of human keratoepithelin in BL21 (DE3)

Keratoepithelin (KE) is an extracellular protein participating in cell adhesion and differentiation. Mutations of the KE gene (on 5q31 in humans) cause deposition of abnormal proteins (amyloid and non-amyloid) in corneal stroma and lead to several corneal dystrophies in humans. However, further studies on the KE protein have been limited by the intrinsic difficulty of purifying this protein. A high-expression plasmid containing human KE gene was constructed to generate recombinant KE proteins in Escherichia coli. The plasmid was transformed into E. coli BL21 (DE3) and the recombinant protein was expressed as an insoluble His-tagged fusion protein and purified by nickel chelation affinity chromatography under denaturing conditions. On average, 12 mg of purified KE was routinely obtained from 1L of culture media. The recombinant KE was refolded in arginine-containing dialysis solutions and the recovery of bioactive KE typically was approximately 70%. The procedures developed in this report should enable reproducible production of KE and related mutant proteins in large quantities and facilitate future studies on biochemical and biophysical properties of KE and the pathogenesis of related corneal dystrophies.     

Conformational changes in the extracellular β-lactamase I from Bacillus cereus 569/H/9

1. The thermal denaturation and precipitation of beta-lactamase I from Bacillus cereus 569/H/9 at 60 degrees C are reversible, a soluble and almost fully active enzyme being obtained after solution of the precipitate in 5m-guanidinium chloride or 8m-urea and subsequent removal of the denaturing agent. 2. Inactivation of beta-lactamase I occurs rapidly between 50 degrees and 55 degrees C and is shown by circular-dichroism spectra to be accompanied by an extensive conformational change. 3. A change to a different conformation occurs in 6m-urea. This change is also reversible; refolding with almost complete recovery of enzymic activity occurs within 5min of dilution of the denaturing agent. 4. Inactivation of beta-lactamase I at pH3.0 and 11.0 is also associated with conformational changes, since a proportion of the lost activity is recovered within 5min of adjustment of the pH to 7.0.     

Synthesis and physicochemical properties in aqueous solution of the sequential polypeptide poly(Tyr-Ala-Glu)

A polypeptide having the repealing sequence (Tyr-Ala-Glu)_n was synthesized by the polymerization of the N-hydroxysuccinimide ester of O-benzyl-L -tyrosyl-L -alanyl-γ-benzyl-L -glutamate, followed by the removal of the benzyl groups by means of hydrogen bromide. The main fraction obtained on gel filtration had an average molecular weight of over 60, 000, corresponding to over 500 amino acid residues per polypcptide chain. The polymer is soluble in water above pH 5.5, and precipitates on lowering the pH. The x-ray powder photographs show features of an α-helical structure. The dependence of the ultraviolet absorption spectrum, the optical rotatory dispersion, and the fluorescence of poly(Tyr-Ala-Glu) on pH, in salt-free as well as in salt-containing aqueous solutions, was compared with the corresponding properties of a copolymer containing equimolar proportions of tyrosine, alanine, and glutamic acid in a random sequence. From these measurements it was concluded that poly(Tyr-Ala-Glu ) has a helical con formation at low pH and a random coil conformation at high pH, the transition taking place at pH 6 in the absence of salt and pH II in the presence of salt. Thus, in the range pH 7 to l0. random coil-to-helix transition can be achieved by merely increasing the ionic strength. A model is proposed for the structure of the helical poly peptide which accounts for the Stability of the helical conformation by assuming hydrogen bonding between the carboxylate group of the ith glutamic acid residue and the hydroxyl group of the (i + 4 )th tyrosine residue. The complex ORD of helical poly(Tyr-Ala-Glu) is explained as being due to a superposition of the ORD of an α-helix and that of a regular array of phenolic ehroniopholes originating from the immobilization of the aromatic rings in the specific structure of the polymer.     

Association of putrescine,spermidine, spermine,and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of high-affinity uptake showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg²⁺ did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.     

The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform

An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized. The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes. The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones. The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions. The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment. Plasmids incorporating respective expression cassettes were used to transform the host strain A. adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions. Transformants of both types were found to harbor a single copy of the heterologous DNA. Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4. In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved. This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette. The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.     

Cleaved serpin refolds into the relaxed state via a stressed conformer

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.     

Expression,purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro

In this work, the recombinant human bone morphogenetic protein 2 (rhBMP-2) gene was cloned from MG-63 cells by RT-PCR, and the protein was expressed in Escherichia coli expression system, purified by Ni–NTA column under denaturing conditions and refolded at 4 °C by urea gradient dialysis. We found that the protein refolding yield was increased with the increase of pH value from pH 6.0 to pH 9.0. The yield was 42% and 96% at pH 7.4 and pH 9.0, respectively, while that at pH 6.0 was only 3.4%. The cell culture results showed that the rhBMP-2 refolded at pH 7.4 urea gradient dialysis had higher biological activity for MG-63 cell proliferation and differentiation than that refolded at pH 9.0 since pH 7.4 is closer to the conditions in vivo leading to the formation of dimers through the interchain disulfide bond. Moreover, the biological activity for MG-63 was promoted with the increase of rhBMP-2 concentration in the cell culture medium. This work may be important for the in vitro production and biomedical application of rhBMP-2 protein.