Department of Biology II and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany.
Published: June 2021
AI Article Synopsis
Article Abstract
The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
When constructing cell factories, it is crucial to reallocate intracellular resources towards the synthesis of target compounds. However, imbalanced resource allocation can lead to a tradeoff between cell growth and production, reducing overall efficiency. Reliable gene expression regulation tools are needed to coordinate cell growth and production effectively.
Noncanonical amino acids (ncAAs) provide numerous avenues for the introduction of novel functionality to peptides and proteins. ncAAs can be incorporated through solid-phase synthesis or genetic code expansion in conjugation with heterologous expression of the encoded protein modification. Due to the difficulty of synthesis or overexpression, wide chemical space, and lack of empirically resolved structures, modeling the effects of ncAA mutation is critical for rational protein design.
Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in eukaryotes has predominantly relied on the pyrrolysyl-tRNA synthetase/tRNA pair. However, access to additional easily engineered pairs is crucial for expanding the structural diversity of the ncAA toolbox in eukaryotes. The -derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair presents a particularly promising alternative.
The analysis of protein-bound glycans has gained significant attention due to their pivotal roles in physiological and pathological processes like cell-cell recognition, immune response, and disease progression. Routine methods for glycan analysis are challenged by the very similar physicochemical properties of their carbohydrate components. As an alternative, lectins, which are proteins that specifically bind to glycans, have been integrated into biosensors for glycan detection.
Department of Medical Oncology, State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China; Shaoxing Institute, Zhejiang University, Shaoxing 312099, China. Electronic address:
Despite the important functions of protein post-translational modifications (PTMs) in numerous cellular processes, understanding the biological roles of PTMs remains quite challenging. Here, we summarize our efforts in recent years to incorporate a variety of non-canonical amino acids (ncAAs) to study the biological functions of protein PTMs in mammalian cells, with a focus on the use of ncAA tools to probe the biological functions of various protein PTMs. We design length-tunable lipidation mimics for studying lipidation function and designing protein drugs.
