Biology subjects

Ortega, C.

Publications and source records attributed to Ortega, C..

3 recordsLinked to original sources

bioRxiv2024

Efficient synthesis of arabinosyl nucleosides via active site engineering of the Leishmania mexicana purine 2'-deoxyribosyltranferase

Even though 2'-deoxyribosyltransferases (NDTs) have emerged as promising and eco-friendly catalysts for synthesizing different 2-deoxynucleoside analogs, their limited activity towards substrates featuring modifications at the 2' -C and 3' -C positions of the ribose moiety significantly restrict their potential application in the pharmaceutical industry. Herein we report two engineered variants from Leishmania mexicana purine deoxyribosyltransferase (LmPDT) with the highest reported arabinosyltransferase activity for an NDT, achieved through a semi-rational design approach. The resulting variants, LmPDTN56L and LmPDTN56C, display up to 13.5- and 30-fold enhanced activity on the synthesis of vidarabine compared to the wild-type enzyme. Moreover, thermal unfolding and thermal challenge experiments revealed that the improvement in arabinosyl transferase activity did not result in a stability trade-off, with Tm values similar to the wild-type enzyme and similar activity retention at 40 {degrees}C. After biochemical characterization, the optimal reaction parameters for variant LmPDTN56C were determined to be pH 6 and a temperature interval from 30 to 45 {degrees}C. Interestingly, under optimal operational conditions (40 {degrees}C, 50 mM MES buffer pH 6) the initial 30-fold enhancement observed for LmPDTN56C escalated to a remarkable 40-fold improvement. This comprehensive study elucidates the potential of the engineered LmPDT variants for efficient and tailored biocatalytic synthesis of arabinosyl nucleosides, paving the way for enhanced applications in nucleoside analog production.

biochemistry↗

bioRxiv2024

A versatile reporter system to study cell-to-cell and cell-free bovine leukemia virus infection

Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic retrovirus of the genus Deltaretrovirus that infects dairy cattle worldwide and is the causative agent of enzootic bovine leukosis. BLV demonstrates remarkably low efficiency in infecting cells via free viral particles derived from infected B cells, as virions are rarely detected in the bloodstream of infected cattle. However, transmission efficacy significantly increases upon the establishment of direct cell-to-cell interactions. Syncytium formation assays are the main tool in the study of BLV infectivity. Although this traditional method is highly robust, the complexity of visually counting syncytia poses a significant technical challenge. Using lentiviral vectors, we generated a stable reporter cell line, in which the GFP reporter gene is under the control of the full-length BLV-LTR. We have demonstrated that the BLV-Tax protein and histone deacetylases inhibitors, such as VPA and TSA, can transactivate the BLV LTR. Upon co-culturing the reporter cell line with BLV-infected cells, fluorescent syncytia can be visualized. By implementing automated scanning and image acquisition using a confocal microscope, together with the development of an analysis software, we can detect and measure single GFP cells and fluorescent multinucleated cells. In summary, our reporter cell line, combined with the development of analysis software, is a useful tool for understanding the role of cell fusion and cell-free mechanism of transmission in BLV infection.

microbiology↗

bioRxiv2019

High-performance chemical and light-inducible recombinases in mammalian cells and mice

Site-specific DNA recombinases are some of the most powerful genome engineering tools in biology. Chemical and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, the availability of inducible recombinases is scarce due to the challenge of engineering high performance systems with low basal activity and sufficient dynamic range. This limitation constrains the sophistication of genetic circuits and animal models that can be created. To expand the number of available inducible recombinases, here we present a library of >20 orthogonal split recombinases that can be inducibly dimerized and activated by various small molecules, light, and temperature in mammalian cells and mice.\n\nFurthermore, we have engineered inducible split Cre systems with better performance than existing inducible Cre systems. Using our orthogonal inducible recombinases, we created a \"genetic switchboard\" that can independently regulate the expression of 3 different cytokines in the same cell. To demonstrate novel capability with our split recombinases, we created a tripartite inducible Flp and a 4-Input AND gate. We have performed extensive quantitative characterization of the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs in terms of signal-to-noise ratio (SNR). To facilitate sharing of this set of reagents, we have deposited our library to Addgene. This library thus significantly expands capabilities for precise and multiplexed mammalian gene expression control.

synthetic biology↗