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Biology subjects

Aranko, A. S.

Publications and source records attributed to Aranko, A. S..

4 recordsLinked to original sources

Engineering the bacterial cellulose-forming surface as a programmable protein recruitment interface

Synthetic biology has advanced microorganisms to be programmed as production hosts, but its application to bacteria that inherently assemble extracellular materials remains limited. Komagataeibacter spp., natively synthesizes cellulose at the bacterial cell surface, creating a material-forming interface that has not been used as a programmable recruitment platform. Here we establish cell-surface display in Komagataeibacter intermedius and show that this interface can recruit defined proteins, making functionalization part of cellulose formation. By engineering LppOmpA, we displayed a fluorescent protein and genetically encoded capture modules (SpyTag and SilkTag) to selectively capture catcher-fused protein cargos onto K. intermedius cell surface. Recruitment of silk-derived structural protein before cellulose production generated silk-associated fibrous structures within the pellicles, with retained cargo signal after washing. The resulting biocomposite showed reorganized fibre-network morphology, increased surface hydrophobicity, mesoscale ordering, and improved wet-state compressive strength. Wild-type cells exposed to same conditions did not reproduce these changes, demonstrating that material properties arise from surface-directed recruitment rather than protein exposure alone. This work demonstrates the material-forming bacterial surface as a programmable engineering interface for organizing extracellular proteins, providing a general strategy for engineering living materials.

bioengineering↗

Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

Protein/peptide pairs, called Catcher/Tag pairs, are applied for biological isopeptide-bond mediated Click-reactions. Covalent protein ligation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines. It is essential to increase the current toolbox of Catcher/Tag pairs to expand the range of applications further, e.g., for multiple-fragment ligation that requires several orthogonal ligases. We report here engineering of novel Catcher/Tag pairs for protein ligation, aided by a new crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We engineer several split variants, characterize in detail one of them, named SilkCatcher/Tag pair, and show that the newly engineered SilkCatcher/Tag pair is orthogonal and compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the new SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90% purity, which is not possible with the traditional recombinant production.

biochemistry↗

Convergent evolution of the Hedgehog/Intein fold in protein splicing

The widely used molecular evolutionary clock assumes the divergent evolution of proteins. Convergent evolution has been proposed only for small protein elements but not for an entire protein fold. We investigated the structural basis of the protein splicing mechanism by class 3 inteins, which is distinct from class 1 and 2 inteins. We gathered structural and mechanistic evidence supporting the notion that the Hedgehog/INTein (HINT) superfamily fold, commonly found in protein splicing and related phenomena, could be an example of convergent evolution of an entire protein fold. We propose that the HINT fold is a structural and biochemical solution for trans-peptidyl and trans-esterification reactions.

biochemistry↗

SUMO-based expression and purification of dermcidin-derived DCD-1L, a human antimicrobial peptide, in Escherichia coli

The dermcidin-derived peptide DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. Thus, it offers a promising alternative to conventional antibiotics. Furthermore, it plays a role in wound healing, atopic dermatitis and acne vulgaris, indicating applications in cosmetic industries. Recently, dermcidin has been identified as a tumor marker improving cancer prognosis. Hence, large quantities of purified DCD-1L peptide are required to meet the needs of basic research and clinical trials. In the current study, we demonstrate SUMO-based heterologous DCD-1L production in Escherichia coli, followed by affinity chromatography purification. The SUMO tag is cleaved with SUMO-specific protease following purification, leaving free DCD-1L peptide without any additional amino acids. The mass of the peptide was further confirmed by MALDI-TOF-TOF analysis. Furthermore, the cleaved DCD-1L showed antimicrobial activity against the E. coli DH5 alpha test strain. The production and purification of DCD-1L using SUMO tag compare advantageously to other protocols previously described. Thus, the SUMO tag system enables large scale recombinant production of the antimicrobial peptide DCD-1L, which constitutes pharmaceutical and therapeutic potential as an alternative antibiotic.

molecular biology↗