Using High Pressure and Alkaline pH for Refolding.

The expression of recombinant proteins as insoluble inclusion bodies (IB) has the advantage to separate insoluble aggregates from soluble bacterial molecules, thus obtaining proteins with a high degree of purity. Even aggregated, the proteins in IB often present native-like secondary and tertiary structures, which can be maintained as long as solubilization is carried out in non-denaturing condition. High pressure solubilizes IB by weakening hydrophobic interactions, while alkaline pH solubilizes aggregates by electrostatic repulsion. The combination of high pressure and alkaline pH is effective for IB solubilization at a mild, non-denaturing condition, which is useful for subsequent refolding. Here, we describe the expression of recombinant proteins in Escherichia coli using a rich medium to obtain high expression levels, bacterial lysis, and washing of the IB to obtain products of high purity, and, finally, the solubilization and high yield of refolded proteins using high pressure and alkaline pH.

Human growth hormone inclusion bodies present native-like secondary and tertiary structures which can be preserved by mild solubilization for refolding.

BACKGROUND: Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. RESULTS: In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield). CONCLUSIONS: We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.

High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: Application of high pressure at pH 9.0.

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.

Refolding of metacaspase 5 from Trypanosoma cruzi, structural characterization and the influence of c-terminal in protein recombinant production.

Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several parasites that cause diseases of great interest to public health, mostly without adequate treatment and included in the neglected tropical diseases category. One of them is Trypanosoma cruzi which causes Chagas disease and has two metacaspases involved in its PCD: TcMCA3 and TcMCA5. Their roles seemed different in PCD, TcMCA5 appears as a proapoptotic protein negatively regulated by its C-terminal sequence, while TcMCA3 is described as a cell cycle regulator. Despite this, the precise role of TcMCA3 and TcMCA5 and their atomic structures remain elusive. Therefore, developing methodologies to allow investigations of those metacaspases is relevant. Herein, we produced full-length and truncated versions of TcMCA5 and applied different strategies for their folded recombinant production from E. coli inclusion bodies. Biophysical assays probed the efficacy of the production method in providing a high yield of folded recombinant TcMCA5. Moreover, we modeled the TcMCA5 protein structure using experimental restraints obtained by XLMS. The experimental design for novel methods and the final protocol provided here can guide studies with other metacaspases. The production of TcMCA5 allows further investigations as protein crystallography, HTS drug discovery to create potential therapeutic in the treatment of Chagas' disease and in the way to clarify how the PCD works in the parasite.

Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli.

Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.

Protein refolding based on high hydrostatic pressure and alkaline pH: Application on a recombinant dengue virus NS1 protein.

In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.

Refolding and Activation from Bacterial Inclusion Bodies of Trypsin I from Sardine (Sardinops sagax caerulea).

BACKGROUND: Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. METHODS: TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. RESULTS: Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. CONCLUSION: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.

Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus.

BACKGROUND: Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. RESULTS: Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. CONCLUSIONS: The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.

Purification and characterization of a lectin with refolding ability from Genipa americana bark.

Genipa americana L., commonly known as genipap, is a plant with economical and medicinal importance, and a promising source of bioactive compounds. Lectins are carbohydrate-binding proteins with several biotechnological applications. This study reports the isolation and characterization of a G. americana bark lectin (GaBL). A single chromatographic procedure on Sephacryl S-100 resulted in isolation of GaBL, a protein with native molecular weight of over 200 kDa and pI 4.02, whose hemagglutinating activity was inhibited by lactose and fetuin, not affected by ions (Ca2+ and Mg2+), and stable upon heating (303-393 K) as well as over the pH range 5-10. The highest activity was found at a temperature lower than 333 K and pH 5. The secondary structure was analyzed by circular dichroism and showed a prevalence of beta structures and unordered forms. GaBL was able to partially refold in acidic pH conditions when dissolved in PBS buffer at pH 7.4. In conclusion, GaBL was purified in milligram quantities with high stability against different conditions, and is a new biomaterial with potential biotechnological applications.

An improved method for purification and refolding of recombinant HIV Vif expressed in Escherichia coli.

Virion infectivity factor (Vif) is a 23 kDa protein that protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the elongin B and C subunits, Cullin 5, and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein. For that purpose, we have cloned Vif in the pET28a expression vector, expressing the resulting His-tagged recombinant protein in the BL21(DE3) Escherichia coli strain. After lysis, Vif was solubilized from the insoluble fraction with 6 M guanidinium chloride and purified by denaturing immobilized-metal affinity chromatography, refolding the protein afterwards by dialysis. The use of 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.2 and the presence of EDTA improved Vif refolding yields by reducing the formation of insoluble aggregates. The purified protein was bound by two monoclonal antibodies against sequential and conformational epitopes located at the C and N terminus, respectively.

Analysis of the carbohydrate-binding-module from Fragaria x ananassa α-L-arabinofuranosidase 1.

α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.

Expresión y renaturalización del antígeno protector de Bacillus anthracis: un modelo para evaluar el replegado de proteínas antigénicas recombinantes a gran escala / Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding

Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.

El antígeno protector de Bacillus anthracis (protective antigen, PA) es una importante proteína inmunogénica, en la que se basan tanto las vacunas contra el ántrax/carbunclo como varios métodos diagnósticos. Para estas aplicaciones es esencial que el PA mantenga sus propiedades antigénicas, para lo cual debe estar correctamente plegado. En este estudio se obtuvieron altos niveles del PA en Escherichia coli recombinante. Inicialmente, la proteína se acumuló desnaturalizada en cuerpos de inclusión, lo que facilitó su eficiente purificación en simples pasos de lavado, pero no fue reconocida por anticuerpos específicos. Se analizaron las condiciones de replegado con un sistema de alto rendimiento, evaluando simultáneamente casi un centenar de condiciones diferentes. La recuperación de la capacidad del PA de ser reconocido por los anticuerpos se evaluó por dot blot utilizando un coeficiente que proporcionó una medida de los niveles de proteína correctamente plegada, con un alto grado de discriminación. Las mejores condiciones de renaturalización permitieron un aumento de diez veces en la intensidad de los dot blots con respecto del control. El único aditivo que produjo buenos resultados de forma constante fue la L-arginina. El análisis estadístico de las interacciones entre los diferentes aditivos de replegado permitió identificar tanto interacciones cooperativas como negativas. El enfoque de alto rendimiento descripto en este trabajo, que permitió la sobreproducción, purificación y plegado del PA de una manera sencilla y directa, puede ser potencialmente útil para el rápido screening de las condiciones adecuadas de replegado cuando se sobreexpresan otras proteínas antigénicas.

Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.

Complex kinetics and residual structure in the thermal unfolding of yeast triosephosphate isomerase.

BACKGROUND: Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein that shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature. RESULTS: Thermally denatured yTIM showed a clear trend towards the formation of aggregation-prone, ß-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Besides, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. This kinetic complexity is, however, not linked to the appearance of residual structure. Furthermore, the rate constant for the main unfolding phase shows small, rather unvarying values in the pH region where denatured yTIM gradually acquires a ß-strand-like conformation. It appears, therefore, that the residual structure has no influence on the kinetic stability of the native protein. However, the presence of residual structure is clearly associated with increased irreversibility. CONCLUSIONS: The slow temperature-induced unfolding of yeast TIM shows three kinetic phases. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. ß-strand-type residual structure, which appears below pH 8.0, is likely to be associated with increased irreversible aggregation of the unfolded protein. However, this denatured form apparently accelerates the refolding process.

Conformational Heterogeneity Determined by Folding and Oligomer Assembly Routes of the Interferon Response Inhibitor NS1 Protein, Unique to Human Respiratory Syncytial Virus.

The nonstructural NS1 protein is an essential virulence factor of the human respiratory syncytial virus, with a predominant role in the inhibition of the host antiviral innate immune response. This inhibition is mediated by multiple protein-protein interactions and involves the formation of large oligomeric complexes. There is neither a structure nor sequence or functional homologues of this protein, which points to a distinctive mechanism for blocking the interferon response among viruses. The NS1 native monomer follows a simple unfolding kinetics via a nativelike transition state ensemble, with a half-life of 45 min, in agreement with a highly stable core structure at equilibrium. Refolding is a complex process that involves several slowly interconverting species compatible with proline isomerization. However, an ultrafast folding event with a half-life of 0.2 ms is indicative of a highly folding compatible species within the unfolded state ensemble. On the other hand, the oligomeric assembly route from the native monomer, which does not involve unfolding, shows a monodisperse and irreversible end-point species triggered by a mild temperature change, with half-lives of 160 and 26 min at 37 and 47 °C, respectively, and at a low protein concentration (10 µM). A large secondary structure change into ß-sheet structure and the formation of a dimeric nucleus precede polymerization by the sequential addition of monomers at the surprisingly low rate of one monomer every 34 s. The polymerization phase is followed by the binding to thioflavin-T indicative of amyloid-like, albeit soluble, repetitive ß-sheet quaternary structure. The overall process is reversible only up until ~8 min, a time window in which most of the secondary structure change takes place. NS1's multiple binding activities must be accommodated in a few binding interfaces at most, something to be considered remarkable given its small size (15 kDa). Thus, conformational heterogeneity, and in particular oligomer formation, may provide a means of expand its binding repertoire. These equilibria will be determined by variables such as macromolecular crowding, protein-protein interactions, expression levels, turnover, or specific subcellular localization. The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu. The implications of NS1 oligomers in the viral life cycle and the inhibition of host innate immune response remain to be determined.

Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis.

The gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable economical loss. Cold shock proteins are conserved in many bacteria and eukaryotic cells and they help to restore normal cell functions after cold shock in which some appear to have specific functions at normal growth temperature as well. Cold shock protein A from C. pseudotuberculosis was expressed in Escherichia coli and purified. The thermal unfolding/refolding process characterized by circular dichroism, differential scanning calorimetry and NMR spectroscopy techniques indicated that the refolding process was almost completely reversible.

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach.

The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1-800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1mM GSSG, 0.75 M guanidine hydrochloride, and 0.5M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.

The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis.

Trichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like 34kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains the prepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim of this work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specific inhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conformation of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopy and fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistant extracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymograms, the inhibitory effect of ppTvCP4r on trichomonad CP proteolytic activity was observed in the ∼97, 65, 39, and 30 kDa regions. By using 2-D zymograms and mass spectrometry, several of the CPs inhibited by ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like protein spots (TvCP2, TvCP4, TvCP4-like, and TvCP39) was observed compared with the control zymogram. Moreover, pretreatment of live parasites with ppTvCP4r inhibited trichomonal haemolysis in a concentration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor of the proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence properties depending on clan CA papain-like CPs.

VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.