High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.Pubblicat

Baseline Plasma Osteopontin Protein Elevation Predicts Adverse Outcomes in Hospitalized COVID-19 Patients

More than three years have passed since the first case, and COVID-19 is still a health concern, with several open issues such as the lack of reliable predictors of a patient's outcome. Osteopontin (OPN) is involved in inflammatory response to infection and in thrombosis driven by chronic inflammation, thus being a potential biomarker for COVID-19. The aim of the study was to evaluate OPN for predicting negative (death or need of ICU admission) or positive (discharge and/or clinical resolution within the first 14 days of hospitalization) outcome. We enrolled 133 hospitalized, moderate-to-severe COVID-19 patients in a prospective observational study between January and May 2021. Circulating OPN levels were measured by ELISA at admission and at day 7. The results showed a significant correlation between higher plasma concentrations of OPN at hospital admission and a worsening clinical condition. At multivariate analysis, after correction for demographic (age and gender) and variables of disease severity (NEWS2 and PiO2/FiO2), OPN measured at baseline predicted an adverse prognosis with an odds ratio of 1.01 (C.I. 1.0-1.01). At ROC curve analysis, baseline OPN levels higher than 437 ng/mL predicted a severe disease evolution with 53% sensitivity and 83% specificity (area under the curve 0.649, p = 0.011, likelihood ratio of 1.76, (95% confidence interval (CI): 1.35-2.28)). Our data show that OPN levels determined at the admission to hospital wards might represent a promising biomarker for early stratification of patients' COVID-19 severity. Taken together, these results highlight the involvement of OPN in COVID-19 evolution, especially in dysregulated immune response conditions, and the possible use of OPN measurements as a prognostic tool in COVID-19