Fabrication and characterization of fast dissolving glycerol monolaurate microemulsion encapsulated gelatin nanofibers with antimicrobial activity.

BACKGROUND: Electrospun fibers are a good candidate for the delivery of bioactive compounds in the food industry because of their advantages that include a tunable diameter, high porosity and a high specific surface area. In the present study, we fabricated gelatin/glycerol monolaurate (GML) microemulsion nanofibers by solubilizing GML in Tween-80 followed by mixing with gelatin solution for electrospinning. We hypothesized that the addition of GML microemulsions affects the properties of the gelatin solution and modifies the physical and antimicrobial properties of the resulting nanofibers. RESULTS: Both pure gelatin solution and gelatin/GML microemulsions showed shear-thinning behavior. However, electrospinnability was not affected by the addition of GML microemulsions. A significantly higher average diameter of nanofibers (1147 nm) with 5% GML was observed compared to the gelatin fiber diameter of 560 nm. Fourier transform infrared spectroscopy showed hydrogen bonding between gelatin molecules and GML microemulsions. Thermal analysis and X-ray diffraction indicated an amorphous structure of gelatin/GML microemulsion nanofibers, although a small amount of crystalline GML existed in the nanofibers with high GML content. Gelatin/GML microemulsion nanofibers showed high thermal stability and improved hydrophilicity. Nanofibers with 5% GML (weight with respect to nanofiber) (D64 nanofibers) showed effective antimicrobial activity against Escherichia coli and Staphylococcus aureus. CONCLUSION: Gelatin/GML microemulsion nanofibrous films demonstrate superhydrophilicity and fast dissolution properties as a result of the high surface-to-volume ratio, amorphous structure and improved hydrophilicity of the nanofiber surface. The results indicate the potential application of gelatin/GML microemulsion nanofibrous films as edible antimicrobial food packaging. © 2021 Society of Chemical Industry.

Expanding the Structural Diversity of Protein Building Blocks with Noncanonical Amino Acids Biosynthesized from Aromatic Thiols.

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications. To expand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed a general and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols. We demonstrate that nearly 50 ncAAs with a diverse array of structures can be biosynthesized from these simple small-molecule precursors by hijacking the cysteine biosynthetic enzymes, and the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code. Moreover, we demonstrate that bioorthogonal reactive groups such as aromatic azides and aromatic ketones can be incorporated into green fluorescent protein or a therapeutic antibody with high yields, allowing for subsequent chemical conjugation.

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates.

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

Efficient Selection Scheme for Incorporating Noncanonical Amino Acids Into Proteins in Saccharomyces cerevisiae.

With the advances in the field of expanded genetic code, the application of non-canonical amino acid (ncAA) is considered an effective strategy for protein engineering. However, cumbersome and complicated selection schemes limit the extensive application of this technology in Saccharomyces cerevisiae. To address this issue, a simplified selection scheme with confident results was developed and tested in this study. Based on a mutation library derived from Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS), a logic gate in synthetic biology was used to optimize screening procedures. We found that an "and" gate was more suitable than an "or" gate for isolating aminoacyl-tRNA synthetase from S. cerevisiae. The successful incorporation of O-methyltyrosine (OMeY) proved the utility and efficiency of this new selection scheme. After a round of positive selection, several new OMeY-tRNA synthetase (OMeYRS) mutants were screened, and their incorporation efficiency was improved. Furthermore, we characterized the insertion of several tyrosine analogs into Herceptine Fab and discovered that OMeYRS and its mutants were polyspecific. One of these mutants showed an optimal performance to incorporate different ncAAs into recombinant proteins in S. cerevisiae; this mutant was cloned and transfected into mammalian cells, and the results proved its functionality in HEK293 cells. This study could expand the application of ncAA in S. cerevisiae to construct efficient yeast cell factories for producing natural and synthetic products.

Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates.

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9-mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9-based genome-editing systems.