Biology subjects

Helalat, S. H.

Publications and source records attributed to Helalat, S. H..

3 recordsLinked to original sources

bioRxiv2024

Sortase A-based Post-Translational Modifications on Encapsulin Nanocompartments

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the biotechnology and nanomedicine area. In this investigation, we effectively developed a sortase A-mediated protein ligation system in E. coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein (GFP) and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research opens up avenues for further exploration of the applications and engineering possibilities of encapsulins in the rapidly advancing field of nanotechnology.

molecular biology↗

bioRxiv2024

Enzymatic Assembly for CRISPR Split-Cas9 System: The Emergence of a Sortase-based Split-Cas9 Technology

CRISPR-Cas9 has been widely used in scientific research and medical investigations as a pioneering technology. However, challenges such as the large size of the Cas9 sequence and potential off-target effects have impeded its widespread adoption. In response, various alternatives, such as split-Cas9 technology, have emerged. Split-Cas9 systems allow the large Cas9 sequence to be divided into two segments to aid in the delivery of the enzyme. Nevertheless, challenges persist in achieving precise control over the timing and location of Cas9 reassembly and activity to minimize off-target effects. This study presents an enzymatic-based split-Cas9 system, introducing a new approach utilizing the Sortase enzyme for the reconstitution of the full Cas9 protein. The developed method eliminates the need for chemical or physical induction and allows for precise genome editing in specific cells through the utilization of various specific promoters or targeted drug delivery. Experimental validation of the enzymatic system was conducted in E. coli, HEK cells, and Jurkat cells, demonstrating successful assembly and activity of the assembled Cas9 enzyme. In addition, this study explored the incorporation of nuclear localization signals, the evaluation of inducible promoters, and the delivery of the systems components in mRNA or protein form. Furthermore, we investigated the potential of S/MAR minicircle technology instead of viral vectors within the system. Overall, we highlighted the feasibility and utility of the Sortase-based split-Cas9 system to enhance control and efficiency compared to traditional CRISPR-Cas9 approaches. Additionally, this study revealed the potential of using the Sortase enzyme for posttranslational modifications and protein assembly in human cells.

molecular biology↗

bioRxiv2023

Metabolic Engineering on a 3D-Printed Microfluidic Platform: A New Approach for Modular Co-Metabolic pathways

Metabolic engineering of cell factories often requires extensive modification of host cellular machinery, leading to numerous challenges such as metabolic burden, intermediate metabolite toxicity, and inadequate endogenous fluxes. To overcome the limitations, we presented an innovative approach for metabolic engineering, by constructing modular biosynthetic pathways on a 3D-printed microfluidic platform. Several new techniques have been developed, including novel designs of chip configurations, effective methods for enzyme immobilization on printed resins, and proper ways to regenerate cofactors in redox reactions. As a proof of concept, we built xylose consumption and CO2 fixation pathways in the microfluidic chips and successfully demonstrated that the platform was able to convert xylose and enable the rapid growth of Saccharomyces cerevisiae, which otherwise will not grow with xylose as the only carbon source. Overall, the 3D-printed microfluidic platform presents a much simpler and more efficient cell-free strategy for developing modular, optimized biosynthetic pathways.

bioengineering↗