Optimizing HIV-1 protease production in Escherichia coli as fusion protein

<p>Abstract</p> <p>Background</p> <p>Human immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the <it>pol </it>gene is responsible for proteolytic processing of the <it>gag-pol </it>polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations.</p> <p>Results</p> <p>A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in <it>Escherichia coli </it>cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for <it>E. coli </it>expression and to avoid autoproteolysis) and by screening six different <it>E. coli </it>strains and five growth media. The best expression yields were achieved in <it>E. coli </it>BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter.</p> <p>Conclusions</p> <p>By using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.</p

ONE-POT ENZIMATIC DEPOLYMERIZATION OF CELLULOSE IN IONIC LIQUIDS

Green alternatives to fossil-based fuels are very attractive and can be produced from cellulosic materials. Cellulose is the primary product of photosynthesis in plants and has immense importance as a renewable raw material. The production of biofuels starting from cellulose is gaining increasing attention and obviously implies the partial or total hydrolysis of cellulose: enzymatic processes are considered the most promising technology [1]. Cellulases (EC 3.2.1.4) are the enzymes most commonly employed to selectively depolymerize cellulose in buffered aqueous solvents. Because of the very low solubility of cellulose due to its highly organized structure, enzymatic conversions proceed at very slow reaction rates and require the dissolution in a solvent to facilitate the access of cellulases to cellulosic substrates. To improve the yield of fermentable monosaccharides, pretreatments of cellulose, such as thermal, chemical or physical treatment, have been applied to afford a better enzymatic conversion [2]. Ionic liquids (ILs) have been increasingly recognized as excellent solvents for dissolution and pretreatment of cellulose but it was previously reported that ILs induce usually fast enzyme deactivation by protein unfolding [3]. In the present work we present a study on a single-batch, homogeneous phase enzymatic hydrolysis of cellulose using three commercial ILs. We have tested two native proteins from Trichoderma reesei and Humicola insolens and two engineered proteins from T. reesei and Streptomyces sp.. In some cases ILs don’t denature the cellulases used but increase their operational stability as compared to standard buffer solutions and facilitate the dissolution of cellulose. Interestingly, the stability of the four cellulases in the presence of the ILs allows to set-up a procedure lacking of the cellulose pretreatment step. We believe that this strategy could be amenable of scale-up and innovative industrial applications for the efficient one-batch conversion of inexpensive cellulosic materials into derivatives (biofuels, derivatized cellulose, monosaccharides for fine chemicals, etc.) with high potential commercial interest and in the framework of environmentally friendly chemistry. References [1] A.P. dadi, S. Varanasi, C.A. Schall. Biotechnol Bioeng, 95(5), 904-910, (2006). [2] M.B. Turner, S.K. Spear, J.G. Huddleston, J.D. Holbrey, R.D. Rogers. Green Chem, 5(4), 443-447, (2003). [3] S.D. Zhu, Y.X. Wu, Q.M. Chen, C. Wang, S. Jin, Y. Ding, G. Wu, Green Chem, 8, 325-327, (2006)

The symmetric active site of enantiospecific enzymes

Biomolecules are frequently chiral compounds, existing in enantiomeric forms. Amino acids represent a meaningful example of chiral biological molecules. Both L- and D-amino acids play key roles in the biochemical structure and metabolic processes of living organisms, from bacteria to mammals. In this review, we explore the enantiospecific interaction between proteins and chiral amino acids, introducing theoretical models and describing the molecular basis of the ability of some of the most important enzymes involved in the metabolism of amino acids (i.e., amino acid oxidases, dehydrogenases, and aminotransferases) to discriminate the opposite enantiomers. Our analysis showcases the power of natural evolution in shaping biological processes. Accordingly, the importance of amino acids spurred nature to evolve strictly enantioselective enzymes both through divergent evolution, starting from a common ancestral protein, or through convergent evolution, starting from different scaffolds: intriguingly, the active sites of these enzymes are frequently related by a mirror symmetry

Lignin valorization: production of high value-added compounds by engineered microorganisms

Lignin is the second most abundant polymer in nature, which is also widely generated during biomass fractionation in lignocellulose biorefineries. At present, most of technical lignin is simply burnt for energy supply although it represents the richest natural source of aromatics, and thus it is a promising feedstock for generation of value-added compounds. Lignin is heterogeneous in composition and recalcitrant to degradation, with this substantially hampering its use. Notably, microbes have evolved particular enzymes and specialized metabolic pathways to degrade this polymer and metabolize its various aromatic components. In recent years, novel pathways have been designed allowing to establish engineered microbial cell factories able to efficiently funnel the lignin degradation products into few metabolic intermediates, representing suitable starting points for the synthesis of a variety of valuable molecules. This review focuses on recent success cases (at the laboratory/pilot scale) based on systems metabolic engineering studies aimed at generating value-added and specialty chemicals, with much emphasis on the production of cis,cis-muconic acid, a building block of recognized industrial value for the synthesis of plastic materials. The upgrade of this global waste stream promises a sustainable product portfolio, which will become an industrial reality when economic issues related to process scale up will be tackled

Biochemical Properties and Physiological Functions of pLG72: Twenty Years of Investigations

In 2002, the novel human gene G72 was associated with schizophrenia susceptibility. This gene encodes a small protein of 153 amino acids, named pLG72, which represents a rare case of primate-specific protein. In particular, the rs2391191 single nucleotide polymorphism (resulting in in the R30K substitution) was robustly associated to schizophrenia and bipolar disorder. In this review, we aim to summarize the results of 20 years of biochemical investigations on pLG72. The main known role of pLG72 is related to its ability to bind and inactivate the flavoenzyme d-amino acid oxidase, i.e., the enzyme that controls the catabolism of d-serine, the main NMDA receptor coagonist in the brain. pLG72 was proposed to target the cytosolic form of d-amino acid oxidase for degradation, preserving d-serine and protecting the cell from oxidative stress generated by hydrogen peroxide produced by the flavoenzyme reaction. Anyway, pLG72 seems to play additional roles, such as affecting mitochondrial functions. The level of pLG72 in the human body is still a controversial issue because of its low expression and challenging detection. Anyway, the intriguing hypothesis that pLG72 level in blood could represent a suitable marker of Alzheimer's disease progression (a suggestion not sufficiently established yet) merits further investigations

Studies on the reaction mechanism of Rhodotorula gracilis D-amino-acid oxidase. Role of the highly conserved Tyr-223 on substrate binding and catalysis.

We have studied D-amino-acid oxidase from Rhodotorula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis. Tyr-223, one of the very few conserved residues among D-amino-acid oxidases, has been mutated to phenylalanine and to serine. Both mutants are active catalysts in turnover with D-alanine, and they are reduced by D-alanine slightly faster than wild-type enzyme. The Tyr-223 --> Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 --> Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active-site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis

Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders

A METABOLIC-LIKE CYCLE FOR SYNTHETIC APPLICATIONS

Systems Biocatalysis is a new approach consisting of organizing enzymes in vitro to generate an artificial metabolism for synthetic purposes. The interconversion of functional groups is the main objective of biocatalysis, and systems organizing a series of enzymes to achieve a multi-step reaction have been reported. The assembly of essentially the same enzymes utilized in Nature to drive the transformation of carbohydrates towards useful synthetic intermediates [1] has been referred to as an artificial metabolism. SysBiocat aims at a similar goal addressing the generalization and organization of group of enzymes (a tool-box) able to perform a series of reactions of general synthetic utility where the feasibility is connected with the obtainment of enzymes of wide substrate specificity or in a rich array of variable common catalytic functions. [2] As a demonstration of this concept, we have recently assembled a biochemical like cycle (Asp-cycle) connecting among them an unsaturated carboxylate (fumaric acid), an alpha-amino acid (L-aspartic acid), a keto acid (oxalacetic acid) and the corresponding alpha-hydroxyacid (D- or L-malic acid). [3] In this view, the obtained cycle may be exploited by coupling it with synthetically relevant reactions which are driven to completion thanks to one or more irreversible steps in the reaction sequence. ____ [1] W.D. Fessner, C. Walter, “Artificial metabolism”, Angew Chem Int Ed, 1992, 31, p. 614 [2] U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K. Robins, “Engineering The Third Wave Of Biocatalysis”, Nature, 2012, 485, p. 185 [3] D. Tessaro, L. Pollegioni, L. Piubelli, P. D’Arrigo, S. Servi, “Systems Biocatalysis: An Artificial Metabolism for Interconversion of Functional Groups”, ACS Catalysis, 2015, 5, p. 160

Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in <it>Escherichia coli </it>of type II cholesterol oxidase from <it>Brevibacterium sterolicum </it>(BCO).</p> <p>Results</p> <p>Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant <it>E. coli </it>cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies.</p> <p>Conclusions</p> <p>Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in <it>E. coli </it>or <it>Streptomyces </it>spp.), shows that the fully active type II BCO can be produced in <it>E. coli </it>at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.</p

Enhancement of the Enzymatic Biosensor Response through Targeted Electrode Surface Roughness

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