Saurabh Gautam

A Novel α-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf) from Clostridium thermocellum

The study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding α-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50°C. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg−1 and 5.0 Umg−1, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of α-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both p-nitrophenol-α-L-arabinofuranoside and p-nitrophenol-α-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% β-sheets, 49% random coils but only 3% α-helices.

Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study

The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

A facile and green ultrasonic-assisted synthesis of BSA conjugated silver nanoparticles.

The formation and growth of hybrid nanoparticles of a protein BSA and silver by ultrasonic assistance were tracked by surface plasmon resonance signal of silver nanoparticles and light scattering. The hybrid nanoparticles were characterized by surface plasmon resonance spectra, light scattering, TEM, circular dichroism spectroscopy and zeta potential. Size along with the spherical shape of the nanoparticles could be controlled and nanoparticles with diameters ranging from 8 to 140 nm could be obtained, depending upon the ultrasonication time (15-30 min) and molar ratio of AgNO(3)/BSA (20-200). The role of single free thiol group in the reduction of silver ions was also investigated by using DTNB modified BSA and protein conjugated silver nanoparticles were formed even with thiol modified BSA. The growth and size of the nanoparticles were governed by ultrasonic assisted Ostwald ripening. BSA conjugated with silver nanoparticles showed changes in the secondary structure with an increase in the beta sheet structure to 33% as compared to 7% in native BSA as determined by CD spectra. Zeta potential measurements in the pH range of 2.0-12.0 demonstrated that the surface charges of the BSA conjugated silver nanoparticles were similar to that of native BSA suggesting that surface charges and overall three dimensional structure of BSA did not change much. This approach provides a strategy for completely green synthesis of hybrid nanoparticles consisting of a biological entity and an inorganic material. This is the first application of ultrasonic assistance in formation of such hybrid nanomaterials in aqueous media.

β-cyclodextrin and curcumin, a potent cocktail for disaggregating and/or inhibiting amyloids: a case study with α-synuclein.

Aggregation of α-synuclein has been implicated in Parkinsons disease (PD). While many compounds are known to inhibit α-synuclein aggregation, dissolution of aggregates into their constituent monomers cannot be readily achieved. In this study, using a range of techniques, we have shown that an optimized cocktail of curcumin and β-cyclodextrin, at appreciably low concentrations, not only inhibited aggregation but also broke up the preformed aggregates almost completely. We propose that these compounds exhibit synergy in their action and thus provide us with the exciting prospect of working toward the development of a suitable drug candidate for prevention and treatment of PD.

Smart polymer mediated purification and recovery of active proteins from inclusion bodies

Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340 nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8 mg L(-1) (for CD4D12, the first two domains of human CD4) to 58 mg L(-1) (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseudochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50 min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (T(m)), and surface hydrophobicity measurement by ANS (8-anilino1-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding.

Polyphenols in combination with β-cyclodextrin can inhibit and disaggregate α-synuclein amyloids under cell mimicking conditions: A promising therapeutic alternative

Parkinsons disease is characterized by the presence of insoluble and neurotoxic aggregates (amyloid fibrils) of an intrinsically disordered protein α-synuclein. In this study we have examined the effects of four naturally occurring polyphenols in combination with β-cyclodextrin (β-CD) on the aggregation of α-synuclein in the presence of macromolecular crowding agents. Our results reveal that even at sub-stoichiometric concentrations of the individual components, the polyphenol-β-CD combination(s) not only inhibited the aggregation of the proteins but was also effective in disaggregating preformed fibrils. Curcumin was found to be the most efficient, followed by baicalein with (-)-epigallocatechin gallate and resveratrol coming in next, the latter two exhibiting very similar effects. Our results suggest that the efficiency of curcumin results from a balanced composition of the phenolic OH groups, benzene rings and flexibility. The latter ensures proper positioning of the functional groups to maximize the underlying interactions with both the monomeric form of α-synuclein and its aggregates. The uniqueness of β-CD was reinforced by the observation that none of the other cyclodextrin variants [α-CD and HP-β-CD] used was as effective, in spite of these possessing better water solubility. Moreover, the fact that the combinations remained effective under conditions of macromolecular crowding suggests that these have the potential to be developed into viable drug compositions in the near future. MTT assays on cell viability independently confirmed this hypothesis wherein these combinations (and the polyphenols alone too) appreciably impeded the toxicity of the prefibrillar α-synuclein aggregates on the mouse neuroblastoma cell lines (N2a cells).

Dendrons and dendrimers as pseudochaperonins for refolding of proteins

Peptide dendrimers are screened for “artificial chaperone” (protein refolding) activity by a sensitive fluorescence based assay. The refolding with largest dendrimer is found to help in recovering biological activity of >90% in the case of unfolded lipases and amylases. The refolding yields decrease down to 14% with a decrease in the complexity and hydrophobicity of the dendron/dendrimer. CD spectroscopy confirms the correct refolding in terms of secondary structure contents of the proteins. The DLS data indicates that presence of the dendrons/dendrimers facilitates protein refolding by preventing the aggregation of proteins.

Simultaneous refolding and purification of recombinant proteins by macro-(affinity ligand) facilitated three-phase partitioning

A strategy called macro-(affinity ligand) facilitated three-phase partitioning (MLFTPP) is described for refolding of a diverse set of recombinant proteins starting from the solubilized inclusion bodies. It essentially consists of: (i) binding of the protein with a suitable smart polymer and (ii) precipitating the polymer-protein complex as an interfacial layer by mixing in a suitable amount of ammonium sulfate and t-butanol. Smart polymers are stimuli-responsive polymers that become insoluble on the application of a suitable stimulus (e.g., a change in the temperature, pH, or concentration of a chemical species such as Ca(2+) or K(+)). The MLFTPP process required approximately 10min, and the refolded proteins were found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The folded proteins were characterized by fluorescence emission spectroscopy, circular dichroism spectroscopy, biological activity, melting temperature, and surface hydrophobicity measurements by 8-anilino-1-naphthalenesulfonate fluorescence. Two refolded antibody fragments were also characterized by measuring K(D) by Biacore by using immobilized HIV-1 gp120. The data demonstrate that MLFTPP is a rapid and convenient procedure for refolding a variety of proteins from inclusion bodies at high concentration. Although establishing the generic nature of the approach would require wider trials by different groups, its success with the diverse kinds of proteins tried so far appears to be promising.

Unusual effects of crowders on heme retention in myoglobin

Myoglobin (Mb) undergoes pronounced heme loss under denaturing conditions wherein the proximal histidine gets protonated. Our data show that macromolecular crowding agents (both synthetic and protein based) can appreciably influence the extent of heme retention in Mb. Interestingly, glucose and sucrose, the monomeric constituents of dextran and ficoll‐based crowders were much more effective in preventing heme dissociation of Mb, albeit, at much higher concentrations. The protein crowders BSA and lysozyme show very interesting results with BSA bringing about the maximum heme retention amongst all the crowding agents used while lysozyme induced heme dissociation even in the native state of Mb. The stark difference that these protein crowders exhibit when interacting with the heme protein is a testament to the varied interaction potentials that a test protein might be exposed to in the physiological (crowded) milieu.

Microwave assisted solubilization of inclusion bodies

BackgroundProduction of recombinant proteins in bacterial hosts often produces insoluble intracellular particles called inclusion bodies. Recovery of active protein from inclusion bodies generally requires their solubilization in chemical denaturants followed by a refolding strategy. The solubilization is carried out with shaking/stirring and takes several hours.ResultsUsing inclusion bodies of seven diverse kinds of recombinant proteins [mutants of controller of cell division or death protein B (CcdB), human CD4D12, thioredoxin fusion protein (malETrx), mutants of maltose binding protein (MBP), single chain variable fragment (ScFv) b12 and single chain antigen binding fragment (ScFab) b12 (anti-HIV-1)], it is shown that exposure to microwave irradiation (200 W) for 2 min, solubilized these inclusion bodies completely. This was confirmed by data based upon turbidity measurements at 400 nm and dynamic light scattering studies. These solubilized inclusion bodies could be refolded correctly in all the cases by known methods. The refolding was confirmed by fluorescence emission spectra and biological activity studies.ConclusionSolubilization of the inclusion bodies before refolding is a part of protein production processes for several recombinant proteins which are overexpressed in the bacterial host systems. Our results show that microwave assistance can considerably shorten the process time.