Free radicals derived from γ-radiolysis of water and AAPH thermolysis mediate oxidative crosslinking of eGFP involving Tyr-Tyr and Tyr-Cys bonds: the fluorescence of the protein is conserved only towards peroxyl radicals.

The enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis. Results demonstrated that protein crosslinking was the major pathway of modification of eGFP towards these oxidants. As evidenced by HPLC-FLD and UPLC-MS, eGFP crosslinking would occur as consequence of a mixture of pathways including the recombination of two protein radicals, as well as secondary reactions between nucleophilic residues (e.g. lysine, Lys) with protein carbonyls. The first mechanism was supported by detection of dityrosine and cysteine-tyrosine bonds, whilst evidence of formation of protein carbonyls, along with Lys consumption, would suggest the formation and participation of Schiff bases in the crosslinking process. Despite of the degree of oxidative modifications elicited by peroxyl radicals (ROO•) generated from the thermolysis of AAPH, and free radicals generated from γ-radiolysis of water, that were evidenced at amino acidic level, only the highest dose of γ-irradiation (10 kGy) triggered significant changes in the secondary structure of eGFP. These results were accompanied by the complete loss of fluorescence arising from the chromophore unit of eGFP in γ-irradiation-treated samples, whereas it was conserved in ROO•-treated samples. These data have potential biological significance, as this fluorescent protein is widely employed to study interactions between cytosolic proteins; consequently, the formation of fluorescent eGFP dimers could act as artifacts in such experiments.

Cloning and expression of the Bacillus amyloliquefaciens transglutaminase gene in E. coli using a bicistronic vector construction.

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.

Transglutaminases: part I-origins, sources, and biotechnological characteristics.

The transglutaminases form a large family of intracellular and extracellular enzymes that catalyze cross-links between protein molecules. Transglutaminases crosslinking properties are widely applied to various industrial processes, to improve the firmness, viscosity, elasticity, and water-holding capacity of products in the food and pharmaceutical industries. However, the extremely high costs of obtaining transglutaminases from animal sources have prompted scientists to search for new sources of these enzymes. Therefore, research has been focused on producing transglutaminases by microorganisms, which may present wider scope of use, based on enzyme-specific characteristics. In this review, we present an overview of the literature addressing the origins, types, reactions, and general characterizations of this important enzyme family. A second review will deal with transglutaminases applications in the area of food industry, medicine, pharmaceuticals and biomaterials, as well as applications in the textile and leather industries.

Review transglutaminases: part II-industrial applications in food, biotechnology, textiles and leather products.

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.

Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan.

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1O2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H218O and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a Kd of 4.8 µM, while data from ITC studies, yielded a Kd of 0.68 µM as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (1O2) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

The mRNA-bound Proteome of Leishmania mexicana: Novel Genetic Insight into an Ancient Parasite.

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.