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Marder L.S.,Grande Rio University | Marder L.S.,Pontifical Catholic University of Rio Grande do Sul | Lunardi J.,Pontifical Catholic University of Rio Grande do Sul | Lunardi J.,Quatro G Pesquisa and Desenvolvimento LTDA | And 16 more authors.
BMC Biotechnology | Year: 2014

Background: Annexin V, a 35.8 kDa intracellular protein, is a Ca+2- dependent phospholipid binding protein with high affinity to phosphatidylserine (PS), which is a well-known hallmark of apoptosis. Annexin V is a sensitive probe for PS exposure upon the cell membrane, and used for detection of apoptotic cells both in vivo and in vitro. Large-scale production of recombinant human annexin V is worth optimization, because of its wide use in nuclear medicine, radiolabeled with 99mTc, for the evaluation of cancer chemotherapy treatments, and its use in identification of apoptotic cells in histologic studies. Here we describe the high-yield production of a tag-free version of human annexin V recombinant protein by linear fed-batch cultivation in a bioreactor.Results: We cloned the human ANXA5 coding sequence into the pET-30a (+) expression vector and expressed rhANXA5 in batch and fed-batch cultures. Using E. coli BL21 (DE3) in a semi-defined medium at 37°C, pH 7 in fed-batch cultures, we obtained a 45-fold increase in biomass production, respective to shaker cultivations. We developed a single-step protocol for rhANXA5 purification using a strong anion-exchange column (MonoQ HR16/10). Using these procedures, we obtained 28.5 mg of homogeneous, nontagged and biologically functional human annexin V recombinant protein from 3 g wet weight of bacterial cells from bioreactor cultures. The identity and molecular mass of rhANXA5 was confirmed by mass spectrometry. Moreover, the purified rhANXA5 protein was functionally evaluated in a FITC-annexin V binding experiment and the results demonstrated that rhANXA5 detected apoptotic cells similarly to a commercial kit.Conclusions: We describe a new fed-batch method to produce recombinant human annexin V in large scale, which may expand the commercial utilities for rhANXAV to applications such as in vivo imaging studies. © 2014 Marder et al.; licensee BioMed Central Ltd.

Lunardi J.,Grande Rio University | Lunardi J.,Quatro G Pesquisa and Desenvolvimento Ltda. | Borges Martinelli L.K.,Grande Rio University | Raupp A.S.,Grande Rio University | And 12 more authors.
RSC Advances | Year: 2016

HisD-Encoded histidinol dehydrogenase (HisD) catalyzes the two last chemical reactions of the l-histidine biosynthetic pathway, namely the conversion of l-histidinol (l-Hol) to l-histidinaldehyde (l-Hal) and to l-histidine (l-His). The hisD gene product has been shown to be essential for Mycobacterium tuberculosis survival in vitro. Herein, we describe a series of biochemical studies on recombinant Mycobacterium tuberculosis HisD (MtHisD). The synthesis of hydrazones derived from l-histidine yielded inhibitors in the low micromolar range, one of which showed moderate anti-Mtb activity. The compounds described here are, to the best of our knowledge, the first inhibitors of MtHisD activity reported in the literature, and they could become promising candidates for future development. © The Royal Society of Chemistry 2016.

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