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

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

Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker

Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines

Assays of D-Amino Acid Oxidase Activity

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples

D-Amino Acid Oxidase-pLG72 Interaction and D-Serine Modulation

pLG72 is a small, primate-specific protein of 153 amino acids. It is the product of the G72 gene, expressed in testis, spinal cord, and brain. The presence of G72 transcript and pLG72 has recurrently been called into question, however G72 mRNA and pLG72 protein levels were higher in blood and brain of patients with schizophrenia than in healthy controls. On the one hand, the SNP rs2391191 corresponding to the R30K substitution in pLG72 was genetically linked to schizophrenia, reduced thickness of the brain cortex in schizophrenia-affected individuals, and altered memory function. Various lines of evidence indicated that pLG72 is a mitochondrial protein, specifically an extrinsic protein bound on the outer membrane. Over the years, pLG72 was proposed to be involved in different functions: (a) overexpression induces mitochondria fragmentation, increasing the numbers of shorter and more mobile ones which could be delivered faster to regions of intense growth and facilitating the dendritic complexity; (b) it might induce oxidative stress by interacting with methionine-R-sulfoxide reductase B2; and (c) it binds and modulates the activity of FMN-containing oxidoreductase of the respiratory complex I. The main role of this protein, however, is related to its binding to the human flavoenzyme D-amino acid oxidase (hDAAO), i.e., the main catabolic enzyme for D-enantiomer of serine. This D-amino acid is a main endogenous coagonist of the N-methyl-D-aspartate type glutamate receptor (NMDAR) involved in main functions such as synaptic plasticity, learning, memory, and excitotoxicity. For this work, we reviewed the recent literature concerning the hDAAO-pLG72 interaction, focusing on the molecular details of the interaction, the effect of hDAAO function and stability, and the cellular effects, especially on D-serine concentration. The main effects related to the pathological R30K substitution are also reported. We have highlighted the gaps in our knowledge of this human protein as well as the relevance of clarifying the molecular details of hDAAO-pLG72 interaction in order to design molecules to modulate hDAAO activity/stability and thus NMDAR function acting at the D-serine cellular level

The Role of D-Amino Acids in Alzheimer's Disease

Alzheimer's disease (AD), the main cause of dementia worldwide, is characterized by a complex and multifactorial etiology. In large part, excitatory neurotransmission in the central nervous system is mediated by glutamate and its receptors are involved in synaptic plasticity. The N-methyl-D-aspartate (NMDA) receptors, which require the agonist glutamate and a coagonist such as glycine or the D-enantiomer of serine for activation, play a main role here. A second D-amino acid, D-aspartate, acts as agonist of NMDA receptors. D-amino acids, present in low amounts in nature and long considered to be of bacterial origin, have distinctive functions in mammals. In recent years, alterations in physiological levels of various D-amino acids have been linked to various pathological states, ranging from chronic kidney disease to neurological disorders. Actually, the level of NMDA receptor signaling must be balanced to promote neuronal survival and prevent neurodegeneration: this signaling in AD is affected mainly by glutamate availability and modulation of the receptor's functions. Here, we report the experimental findings linking D-serine and D-aspartate, through NMDA receptor modulation, to AD and cognitive functions. Interestingly, AD progression has been also associated with the enzymes related to D-amino acid metabolism as well as with glucose and serine metabolism. Furthermore, the D-serine and D-/total serine ratio in serum have been recently proposed as biomarkers of AD progression. A greater understanding of the role of D-amino acids in excitotoxicity related to the pathogenesis of AD will facilitate novel therapeutic treatments to cure the disease and improve life expectancy