Nickel Ferrite Nanoparticles as an Adsorbent for Immobilized Metal Affinity Chromatography of Proteins.

A simple method of preparing amorphous nickel ferrite nanoparticles of about 5 nm diameter is described. These particles were characterized by dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). The nanoparticles were evaluated for their use as a magnetic material for immobilized metal affinity chromatography (IMAC). The ferrite nanoparticles bound to bovine serum albumin (BSA) and the binding fitted Langmuir isotherm model. A high capacity of 916 mg BSA/g dried nanoparticle was observed. Six proteins (Soybean trypsin inhibitor (STI), lactate dehydrogenase (LDH), papain, catalase, ß-galactosidase and casein) were used and all were found to bind at >90% level (except papain which showed 84% binding). All the proteins except LDH and ß-galactosidase could be eluted with 1 M imidazole and with % activity recovery of >80%. Papain could be purified from its dried crude latex by 5-fold and purified papain showed a single band on SDS-PAGE. These nanoparticles constitute a high capacity and are magnetic material useful for IMAC and do not require any pre-functionalization.

Intrinsic disorder in proteins: Relevance to protein assemblies, drug design and host-pathogen interactions.

Intrinsic disorder in proteins resulting in considerable variation in structure can lead to multiple functions including multi-specificity and diverse pathologies. Protein interfaces can involve disordered regions that assemble through a concerted-fold-and-bind mechanism. The binding involves both enthalpic and entropic gains by exploiting 'hot spots' on the partner and displacing water molecules placed in thermodynamically unfavorable situations. The examples of Rad51-BRCA2 and Artemis-DNA-PKCs/LigIV complexes illustrate this in the context of drug design. This overview tracks the seamless involvement of protein disorder in multi-specificity of biocatalysts, protein assembly formations and host-pathogen interactions, where intrinsic disorder can in Mycobacteria, compensate for genome reduction by carrying out multiple functions and in some RNA viruses facilitate adaption to the host. These present challenging opportunities for designing new drugs and interventions.

Protein promiscuity in drug discovery, drug-repurposing and antibiotic resistance.

Proteins are supposed to bind to their substrates/ligands in a specific manner via their pre-formed binding sites, according to classical biochemistry. In recent years, several types of deviations from this norm have been observed and called promiscuous behavior. Enzymatic promiscuities allow several biochemical functions to be carried out by the same enzyme. The promiscuous activity can also be the origin of "new proteins" via gene duplication. In more recent years, proteins from prokaryotes, eukaryotes and viruses have been found to have intrinsic disorder and lack a preformed binding site. Intrinsic disorder is exploited in regulatory proteins such as those that are involved in transcription and signal transduction. Such proteins function by folding locally while binding to their ligands or interacting with other proteins. These phenomena have also been classified as examples of protein promiscuity and encompass diverse kinds of ligands that can bind to a protein. Given the significant extent of structural homology in many protein families, it is not surprising that ligands also have been found to display promiscuity. Promiscuous behavior of proteins offers both challenges and opportunities to the drug discovery programs such as drug repurposing. Pathogens when exposed to antibiotics exploit protein promiscuity in several ways to develop resistance to the drug. There is increasing evidence now to support that the disorder in proteins is a major tool used by pathogens for virulence and evade drug action by exploiting protein promiscuity. This review provides a holistic view of this multi-faceted phenomenon called protein promiscuity.

Effectiveness of cross-linked enzyme aggregates of cellulolytic enzymes in hydrolyzing wheat straw.

Development of industrially potent cellulolytic enzymes is one of the greatest challenges faced in lignocellulosic feed-stock based bio-refining. In the current work cross-linked enzyme aggregates (CLEAs) of commercial cellulase mix were successfully prepared and their performance to be used as potential industrial enzymes in terms of stability and wheat straw hydrolysis was evaluated. The CLEAs were more stable compared to native enzymes with half-lives being 2.30-, 1.56-, 3.07- and 1.67-fold higher at 70°C for filter paper activity (FPA), endoglucanase, ß-glucosidase and xylanase, respectively. CLEAs retained 77.4% of endoglucanase and 85.9% of xylanase activity after five cycles of hydrolysis of soluble substrates such as carboxymethyl cellulose and xylan, respectively. A maximum saccharification yield of 31.8% by soluble enzymes and 32.9% by CLEAs were obtained when alkali-pretreated wheat straw was subjected to hydrolysis. On repeated batch hydrolysis for five consecutive cycles of 24 h each, the CLEAs showed an overall higher saccharification yield of 43.3% compared to 31.8% with soluble enzymes.

Cross-Linked Enzyme Aggregates for Applications in Aqueous and Nonaqueous Media.

Extensive cross-linking of a precipitate of a protein by a cross-linking reagent (glutaraldehyde has been most commonly used) creates an insoluble enzyme preparation called cross-linked enzyme aggregates (CLEAs). CLEAs show high stability and performance in conventional aqueous as well as nonaqueous media. These are also stable at fairly high temperatures. CLEAs with more than one kind of enzyme activity can be prepared, and such CLEAs are called combi-CLEAs or multipurpose CLEAs. Extent of cross-linking often influences their morphology, stability, activity, and enantioselectivity.

Protein-Coated Microcrystals, Combi-Protein-Coated Microcrystals, and Cross-Linked Protein-Coated Microcrystals of Enzymes for Use in Low-Water Media.

Protein-coated microcrystals (PCMC) are a high-activity preparation of enzymes for use in low-water media. The protocols for the preparation of PCMCs of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. The combi-PCMC concept is useful both for cascade and non-cascade reactions. It can also be beneficial to combine two different specificities of a lipase when the substrate requires it. Combi-PCMC of CALB and Palatase used for the conversion of coffee oil present in spent coffee grounds to biodiesel is described. Cross-linked protein-coated microcrystals (CL-PCMC) in some cases can give better results than PCMC. Protocols for the CLPCMC of Subtilisin Carlsberg and Candida antarctica lipase B (CAL B) are described. A discussion of their applications is also provided.

Structural Modelling, Substrate Binding and Stability Studies of Endopectate Lyase (PL1B) of Family 1 Polysaccharide Lyase from Clostridium thermocellum.

An endo-pectate lyase (PL1B) of family 1 polysaccharide lyase from Clostridium thermocellum was structurally characterized and its stability under chaotropic agent was determined. The putative domain PL1B was identified from the protein sequence ABN53381.1 belonging to superfamily 3 of pectate lyase. Multiple sequence alignment of PL1B with other known pectate lyases revealed the conserved and semi-conserved residues. The secondary structure of PL1B predicted by PsiPred and confirmed by Circular Dichroism showed the presence of 2 α-helices (2.06%), 26 ß-strands (40.54%) and 29 random coils (57.4%). The modelled protein represented right handed parallel ß-helix structure, where three parallel ß-sheets linked by loops coils around to form the ß-helix core. Quality assessment of energy minimized structure by Ramachandran plot displayed 82.8% residues in favoured region. Superposition of PL1B structure with Bsp165-PelA from Bacillus sp. revealed the substrate binding cleft formed by the amino acid residues from the loops and ß-sheet. Molecular dynamic simulation of modelled PL1B structure inferred that it is quite stable and compact. Docking studies identified Asp151, Arg209, Asn234, Arg236, Tyr271 and Ser272 as the key residues of PL1B involved during catalysis. Among them Arg209 is responsible for proton abstraction during ß-elimination. Protein melting studies on PL1B showed that there was 12°C shift of peak from 74 to 86°C in presence of 0.6 mM Ca(2+) ions, showing that they provide stability to the structure. The unfolding of PL1B by GuHCl or Urea by fluorescence study showed that the protein structure is stable and disintegrates at their higher concentrations.

The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ~14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that CtCBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of CtCBM6 and that Ca(2+) ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% ß-strands supporting the predicted structure. CtCBM6 displays a jelly roll ß-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting ß4-ß5 and ß8-ß9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of ß-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.

Lipase catalyzed transesterification of castor oil by straight chain higher alcohols.

Biolubricants from Castor oil were produced enzymatically by transesterification with higher alcohols using a lipase mixture of immobilized Mucor miehei (RMIM) and immobilized Candida antarctica lipase B (Novozym 435) under low water conditions. The conversions were in the range of 80-95% under the optimized conditions.

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

This chapter describes two simple interrelated non-chromatographic methods of protein purification. In the first method, called affinity precipitation, inherent affinity of reversibly soluble-insoluble polymers (also called stimuli-sensitive or smart polymers) is exploited to form an affinity complex in free solution with target protein. The affinity complex is precipitated by a suitable change in the medium. The desired protein is dissociated from the smart polymer. In the second method called macro (affinity ligand)-facilitated three phase partitioning (MLFTPP), the affinity complex is precipitated at an interface between upper t-butanol-rich phase and lower aqueous phase. These three phases are achieved by adding appropriate amounts of ammonium sulfate and t-butanol to the initial crude extract. In the first protocol, sequential MLFTPP is used with two different smart polymers to purify pectinase and cellulase from a single crude preparation. The second protocol illustrates the application of the affinity precipitation in simultaneous purification and refolding of a urea-denatured xylanase.

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 Ca(2+) and Mg(2+) 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 Ca(2+) ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% ß-sheets, 49% random coils but only 3% α-helices.

WITHDRAWN: Smart polymer-coated microplate wells: Applications in protein purification, protein refolding, and sensing of analytes.

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Green synthesis of nanocomposites consisting of silver and protease alpha chymotrypsin.

The synergy of ultrasonication and the exposure to light radiation was found to be necessary in the formation of nanocomposites of silver and a protease alpha chymotrypsin. The reaction was carried out in aqueous medium and the process took just less than 35 min. Ultrasonication alone formed very negligible number of nanoparticles of <100 nm size whereas light alone produced enough number but the size of the particles was >100 nm. The effects of pH (in the range of 3-5, 9-10), ultrasonication time periods (0-30 min), ultrasonication intensity (33-83 W cm(-2)), energy of light radiation (short UV, long UV and Fluorescent light) and time period of exposure (5-60 min) to different light radiations were studied. The formation of nanocomposites under these effects was followed by surface plasmon resonance (SPR) spectra, dynamic light scattering (DLS), transmission electron microscopy (TEM). Ag-chymotrypsin nanocomposites of sizes ranging from 13 to 72 nm were formed using the synergy of ultrasonication and exposure to short UV radiation. Results show that ultrasonication promoted nuclei formation, growth and reduction of polydispersity by Ostwald ripening.

Solid state fluorescence of proteins in high throughput mode and its applications.

A simple method to determine fluorescence emission spectra of proteins in solid state is described. The available commercial accessories can only accommodate solid samples and hence do not allow a direct comparison between fluorescence spectra of a sample in solution and solid state form. Such comparisons are valuable to monitor the changes in protein structure when it is "dried" or immobilized on a solid surface (for biocatalysis or sensor applications). The commercially available accessories also do not allow working in a high throughput mode. We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples. This method works with a 96-well plate format. It enables the comparison of fluorescence spectra of a sample in a solid state with solution spectra, using comparable quantities of protein. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form and the lyophilized powder form. α-Chymotrypsin in solution showed a λ max of 335 nm while a high-activity preparation of the same enzyme for non-aqueous media, known as enzyme precipitated and rinsed with propanol (EPRP), showed an increase in the intensity of the fluorescence emission spectra. However, there was a small red shift of 2 nm (λ max of 337 nm) in contrast to lyophilized powder which showed a λ max of 328 nm. This is due to a difference in the tertiary structure of the protein as well as the microenvironment of aromatic residues between the two preparations. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. The relative fluorescence intensity of lyophilized GFP powder was decreased significantly to 17% as compared to GFP in solution, and showed a red shift of 4 nm in the emission λ max. It was found that fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP was significantly diminished. This indicated the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.

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.

Alpha chymotrypsin coated clusters of Fe3O4 nanoparticles for biocatalysis in low water media.

BACKGROUND: Enzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water) by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe3O4 nanoparticles in n-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe3O4 nanoparticles. These preparations were found to have quite high transesterification activity in low water containing n-octane. RESULTS: Precipitation of alpha chymotrypsin over a stirred suspension of Fe3O4 nanoparticles (3.6 nm diameter) led to the formation of enzyme coated clusters of nanoparticles (ECCNs). These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering). Transmission electron microscopy (TEM), showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous n-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3) showed the highest initial rate of 465 nmol min-1 mg-1protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min-1 mg-1 protein.Circular Dichroism (CD)(with a spinning cell accessory) showed that secondary structure content of the alpha Chymotrypsin in ECCN 3 [15% α-helix, 37% ß-sheet and 48% random coil] was identical to the simple precipitates of alpha chymotrypsin. CONCLUSION: A strategy for obtaining a high activity preparation of alpha chymotrypsin for application in low water media is described. Such high activity biocatalysts are useful in organic synthesis.

Role of smart polymers in protein purification and refolding.

Affinity precipitation is a non-chromatographic method which is useful for purification and refolding of proteins. Quite often, a stimuli-sensitive polymer can be identified which selectively binds to the desired protein. For separation, the protein can be recovered from the precipitate of the protein-smart polymer complex. In case of a refolding experiment, binding of the solubilized protein (in its denatured form) with the polymer leads to the refolding of the protein.

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.

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.

Non-chromatographic strategies for protein refolding.

Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding refers to folding back the proteins after solubilizing/unfolding the misfolded proteins of the inclusion bodies. Protein aggregation, a concentration dependent phenomenon, competes with refolding pathway. The refolding strategies largely aim at reducing aggregation and/or promoting correct folding. This review focuses on non-chromatographic strategies for refolding like dilution, precipitation, three phase partitioning and macro-(affinity ligand) facilitated three phase partitioning. The nanomaterials which disperse well in aqueous buffers are also discussed in the context of facilitating protein refolding. Apart from general results with these methods, the review also covers the use of non-chromatographic methods in protein refolding in the patented literature beyond 2000. The patented literature generally describes use of cocktail of additives which results in increase in refolding yield. Such additives include low concentration of chaotropic agents, redox systems, ions like SO4(2-) and Cl-, amines, carboxylic acids and surfactants. Some novel approaches like use of a "pressure window" or ionic liquids for refolding and immobilized diselenide compounds for ensuring correct -S-S- bonds pairing have also been discussed in various patents. In most of the patented literature, focus naturally has been on refolding in case of pharmaceutical proteins.