Biology subjects

Meineke, B.

Publications and source records attributed to Meineke, B..

4 recordsLinked to original sources

bioRxiv2025

Dual-color expansion microscopy of membrane proteins using bioorthogonal labelling

Site-specific incorporation of non-canonical amino acids (ncAAs) combined with bioorthogonal click chemistry provides a powerful tool for fluorescent protein labeling, overcoming the linkage error inherent to antibody-based probes. In this study, we present the development of dual-color super-resolution imaging utilizing ncAA labeling together with expansion microscopy (ExM). After optimizing the labeling procedures and fluorophore selection, we visualize and resolve the nanoscale distribution of Na,K-ATPase 1 and {beta}1 subunits in expanded HEK 293T cells. We validate our approach by super-resolution STED imaging of ncAA labeled {beta}1 subunit in unexpanded cells. This work establishes a robust framework for multiplexed, high-resolution imaging and suggests that the combination of ncAA labeling with ExM has the potential to push biological imaging toward [A]ngstrom-level resolution.

biophysics↗

bioRxiv2023

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Pyrrolysyine-tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) pairs from methanogenic archaea and engineered bacterial tRNA/aminoacyl-tRNA synthetases (aaRS) pairs are used for site-specific incorporation of noncanonical amino acids (ncAAs) in response to stop codons in mammalian cells. Routinely, ncAA incorporation is achieved by transient expression of the tRNA/aaRS pair leading to heterogeneous suppression. Genomic integration of tRNA/aaRS expression cassettes for more homogenous, adjustable and reproducible levels of protein, containing one or more ncAA, will greatly benefit protein engineering, chemical control and imaging applications in mammalian cells. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal tRNAPyl/PylRS or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore simultaneous suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual fluorophore labeling of a cell surface receptor on live mammalian cells.

cell biology↗

bioRxiv2021

Engineered human induced pluripotent cells enable genetic code expansion in brain organoids

Human induced pluripotent stem cell (hiPSC) technology has revolutionized human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to amber stop codons. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/449576v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4e1fa6org.highwire.dtl.DTLVardef@fff373org.highwire.dtl.DTLVardef@1c839e6org.highwire.dtl.DTLVardef@5150e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2018

Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

synthetic biology↗