Combination of the mutations for improving activity of TEV protease in inclusion bodies.

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.

Host and Bacterial Glycolysis during Chlamydia trachomatis Infection.

The obligate intracellular pathogen Chlamydia trachomatis is the leading cause of noncongenital blindness and causative agent of the most common sexually transmitted infection of bacterial origin. With a reduced genome, C. trachomatis is dependent on its host for survival, in part due to a need for the host cell to compensate for incomplete bacterial metabolic pathways. However, relatively little is known regarding how C. trachomatis is able to hijack host cell metabolism. In this study, we show that two host glycolytic enzymes, aldolase A and pyruvate kinase, as well as lactate dehydrogenase, are enriched at the C. trachomatis inclusion membrane during infection. Inclusion localization was not species specific, since a similar phenotype was observed with C. muridarum Time course experiments showed that the number of positive inclusions increased throughout the developmental cycle. In addition, these host enzymes colocalized to the same inclusion, and their localization did not appear to be dependent on sustained bacterial protein synthesis or on intact host actin, vesicular trafficking, or microtubules. Depletion of the host glycolytic enzyme aldolase A resulted in decreased inclusion size and infectious progeny production, indicating a role for host glycolysis in bacterial growth. Finally, quantitative PCR analysis showed that expression of C. trachomatis glycolytic enzymes inversely correlated with host enzyme localization at the inclusion. We discuss potential mechanisms leading to inclusion localization of host glycolytic enzymes and how it could benefit the bacteria. Altogether, our findings provide further insight into the intricate relationship between host and bacterial metabolism during Chlamydia infection.

Enzyme assembly guided by SPFH-induced functional inclusion bodies for enhanced cascade biocatalysis.

Enzyme clustering into compact agglomerates could accelerate the processing of intermediates to enhance metabolic pathway flux. However, enzyme clustering is still a challenging task due to the lack of universal assembly strategy applicable to all enzymes. Therefore, we proposed an alternative enzyme assembly strategy based on functional inclusion bodies. First, functional inclusion bodies in cells were formed by the fusion expression of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain and enhanced green fluorescent protein, as observed visually and by transmission electron microscopy. The formation of SPFH-induced functional inclusion bodies enhanced intermolecular polymerization as revealed by further analysis combined with Förster resonance energy transfer and bimolecular fluorescent complimentary. Finally, the functional inclusion bodies significantly improved the enzymatic catalysis in living cells, as proven by the examples with whole-cell biocatalysis of phenyllactic acid by Escherichia coli, and the production of N-acetylglucosamine by Bacillus subtilis. Our findings suggest that SPFH-induced functional inclusion bodies can enhance the cascade reaction of enzymes, to serve as a potential universal strategy for the construction of efficient microbial cell factories.

Purification and immobilization of α-amylase in one step by gram-positive enhancer matrix (GEM) particles from the soluble protein and the inclusion body.

Immobilization of the enzyme benefits the catalytic industry a lot. The gram-positive enhancer matrix (GEM) particles could purify and immobilize the recombinant α-amylase in one step without changing the enzymatic character. The enzyme immobilized by GEM particles exhibited good reusability and storage stability. The denaturants dissolved some of the GEM particles and a part of the GEM particles could bear the denaturants. The GEM particles had strong binding ability to the recombination protein with the AcmA tag even when the denaturants existed. The inclusion body was dissolved by urea and then bound by the GEM particles. The GEM particles binding the recombination protein were separated by centrifugation and resuspended in the renaturation solution. GEM particles were recycled by repeating the boiling procedure used in preparing them. The recombination α-amylase without any tag was obtained by digestion and separated via centrifugation. Altogether, our findings suggest that GEM particles have the potential to function as both immobilization and purification materials to bind the soluble recombinant protein with the AcmA tag and the inclusion body dissolved in the denaturants.

Gene cloning, expression in E. coli, and in vitro refolding of a lipase from Proteus sp. NH 2-2 and its application for biodiesel production.

OBJECTIVE: To obtain active lipases for biodiesel production by refolding Proteus sp. lipase inclusion bodies expressed in E. coli. RESULTS: A lipase gene lipPN1 was cloned from Proteus sp. NH 2-2 and expressed in E. coli BL21(DE3). Non-reducing SDS-PAGE revealed that recombinant LipPN1(rLipPN1) were prone to form inclusion bodies as disulfide-linked dimers in E. coli. Site-directed mutagenesis confirmed that Cys85 in LipPN1 was involved in the dimer formation. After optimizing the inclusion body refolding conditions, the maximum lipase activity reached 1662 U/L. The refolded rLipPN1 exhibited highest activity toward p-nitrophenyl butyrate at pH 9.0 and 40 °C. It could be activated by Ca2+ with moderate tolerance to organic solvents. It could also convert soybean oil into biodiesel at a conversion ratio of 91.5%. CONCLUSION: Preventing the formation of disulfide bond could enhance the refolding efficiency of rLipPN1 inclusion bodies.

Optimized upstream and downstream process conditions for the improved production of recombinant human asparaginase (rhASP) from Escherichia coli and its characterization.

The present work elucidates the production of recombinant human asparaginase (rhASP) under optimized fermentation and downstream processes in Escherichia coli. The maximum biomass yield of 6.7 g/L was achieved with fed-batch fermentation. The highest rhASP inclusion bodies recovery yield (91%) was achieved with the optimized lysis conditions. The 8.0 M urea at pH 8.5 has shown efficient solubilization (94%) of rhASP inclusion bodies. The refolding efficiency of rhASP increased at pH 8.5 (84%) and temperature 25°C (86%). The diluted rhASP solution was concentrated and partially purified (92%) using cross flow filtration. A single step ion exchange chromatography is successfully achieved the maximum purity of ≥ 97%. The molecular mass of purified rhASP is confirmed as 34.1 kDa by mass spectrometry. The secondary structure of rhASP is characterized by FT-IR spectroscopy based on the structural elements. Finally, cell proliferative assay of purified rhASP is signifies the similar biological activity over the standard.

Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies.

The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli. Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into ß-sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.

Relationship between mutant Cu/Zn superoxide dismutase 1 maturation and inclusion formation in cell models.

A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In this study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time stamp that distinguishes pre-existing and newly made proteins. In cells that transiently over-express the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu co-factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly made protein, are prone to misfolding and aggregation.

Efficient renaturation of inclusion body proteins denatured by SDS.

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins.

Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome.

BACKGROUND: Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase-named Chi18H8 and belonging to family 18 glycosyl hydrolases-was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 µg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential. RESULTS: We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate. CONCLUSIONS: Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.

Change of the N-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human D-amino acid oxidase as active inclusion bodies.

OBJECTIVES: To optimize the production of active inclusion bodies (IBs) containing human D-amino acid oxidase (hDAAO) in Escherichia coli. RESULTS: The optimized initial codon region combined with the coexpressed rare tRNAs, fusion of each of the N-terminal partners including cellulose-binding module, thioredoxin, glutathione S-transferase and expressivity tag, deletion of the incorporated linker, and improvement of tRNA abundance affected the production and activity for oxidizing D-alanine of the hDAAO in IBs. Compared with the optimized fusion constructs and expression host, IBs yields and activity were increased to 2.6- and 2.8-fold respectively by changing the N-terminal codon bias of the hDAAO. The insoluble hDAAO codon variant displayed the same substrate specificity as the soluble one for oxidizing D-alanine, D-serine and D-aspartic acid. The freshly prepared hDAAO codon variant was used for analyzing the L-serine racemization activity of the bacterially expressed maize serine racemase. CONCLUSIONS: Optimization of the N-terminal codon bias combined with the coexpression of rare tRNAs is a novel and efficient approach to produce active IBs of the hDAAO.

Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.

Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations.

Expression, characterization of a novel nitrilase PpL19 from Pseudomonas psychrotolerans with S-selectivity toward mandelonitrile present in active inclusion bodies.

OBJECTIVES: To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step. RESULTS: A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity. CONCLUSIONS: A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.

Enzyme Engineering for In Situ Immobilization.

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

BACKGROUND: The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. METHODS: Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. RESULTS: Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended ß-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. CONCLUSION: In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Isolation and characterization of a 17-kDa FKBP-type peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum.

Peptidyl-prolyl cis/trans isomerase (PPIase) catalyzes the isomerization of peptide bonds to achieve conformational changes in native folded proteins. An FKBP-type PPIase with an approximate molecular weight of 17kDa was isolated from Vibrio anguillarum O1 and named VaFKBP17. To investigate its biochemical properties, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli. A protease-coupled assay for isomerization activity, using Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate, indicated that the activity of VaFKBP17 was highest at low temperature (5°C) and alkaline conditions (pH 10). The immunosuppressant FK506 inhibited the isomerization activity of VaFKBP17. The chaperone activity of VaFKBP17 was assessed using a citrate synthase thermal aggregation activity assay. To evaluate its ability to catalyze protein refolding, the effect of VaFKBP17 on inclusion bodies was investigated during a dilution process. In this assay, VaFKBP17 was able to assist protein refolding. These results provide evidence that VaFKBP17 possesses chaperone-like activity. The structural homology of VaFKBP17 relative to other known bacterial FKBPs was also examined.

Spontaneously Occurring Formation of Intranuclear and Cytoplasmic Inclusions in Renal Proximal Epithelium Due to Accumulation of D-Amino Acid Oxidase in Wistar Hannover Rats.

Intranuclear and cytoplasmic inclusions in the renal proximal tubular epithelium were observed in nontreated male and female Wistar Hannover rats in a 26-week study (32 weeks of age) and a 104-week study (110 weeks of age). The incidence rates were less than 5% in these two studies. In affected animals, the inclusions were observed in more than 60% of proximal tubular epithelium as various sized (approximately 1-8 µm in diameter) round and eosinophilic materials, but not in distal tubules, Henle's loop, or collecting ducts. Ultrastructurally, inclusions appeared finely granular, homogenous with middle-electron density, and without a limiting membrane. These inclusions were determined to be protein histochemically stained by Azan-Mallory and immunoreactive with an antibody against D-amino acid oxidase (DAO). There was no abnormality in in-life observations or in clinical test values suggestive of renal dysfunction. There were no associated degenerative or inflammatory changes in the kidneys, and no similar inclusions were observed in the other organs. These inclusions are very similar to propiverine hydrochloride (propiverine) and norepinephreine/serotonin reuptake inhibitor-induced inclusions. This is the first report of accumulation of DAO and formation of inclusions occurring spontaneously in rat kidneys. The data are important for toxicological studies using Wistar Hannover rats.

Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli.

Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may represent viable drug targets for major diseases. In depth and rigorous studies of the potential for these proteins to be targets for drug development require sufficient amounts of protease protein that can be used for both experimental and therapeutic purposes. Escherichia coli was widely used to express papain-like cysteine proteases, but most of those proteases are produced in insoluble inclusion bodies that need solubilizing, refolding, purifying and activating. Refolding is the most critical step in the process of generating active cysteine proteases and the current approaches to refolding include dialysis, dilution and chromatography. Purification is mainly achieved by various column chromatography. Finally, the attained refolded proteases are examined regarding their protease structures and activities.

Reversible acetylation regulates salt-inducible kinase (SIK2) and its function in autophagy.

Salt-inducible kinase 2 (SIK2) is a serine/threonine protein kinase belonging to the AMP-activated protein kinase (AMPK) family. SIK2 has been shown to function in the insulin-signaling pathway during adipocyte differentiation and to modulate CREB-mediated gene expression in response to hormones and nutrients. However, molecular mechanisms underlying the regulation of SIK2 kinase activity remains largely elusive. Here we report a dynamic, post-translational regulation of its kinase activity that is coordinated by an acetylation-deacetylation switch, p300/CBP-mediated Lys-53 acetylation inhibits SIK2 kinase activity, whereas HDAC6-mediated deacetylation restores the activity. Interestingly, overexpression of acetylation-mimetic mutant of SIK2 (SIK2-K53Q), but not the nonacetylatable K53R variant, resulted in accumulation of autophagosomes. Further consistent with a role in autophagy, knockdown of SIK2 abrogated autophagosome and lysosome fusion. Consequently, SIK2 and its kinase activity are indispensable for the removal of TDP-43Δ inclusion bodies. Our findings uncover SIK2 as a critical determinant in autophagy progression and further suggest a mechanism in which the interplay among kinase and deacetylase activities contributes to cellular protein pool homeostasis.