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Using CFPS to perform site-specific insertion of nnAA, we ingeniously achieve site‑specific PTMs.
Release date:
2024-10-17
Post-translational modifications (PTMs) of proteins greatly expand the diversity of the proteome and are essential for protein biological functions. Common types of PTMs include phosphorylation, glycosylation, acetylation, ubiquitination, and methylation, among others.
When investigating the structure and function of PTMs, it is often necessary to obtain proteins that are homogeneous and modified at specific sites—this frequently represents a major challenge in the field. The reasons for this include:
1. For many modifications identified in large-scale screening, the native enzymes responsible for these modifications in vivo remain unknown.
2. Natural enzymes exhibit limited controllability; some modifying enzymes possess broad substrate specificity, making it difficult to introduce the desired post-translational modification (PTM) at a specific site without affecting other sites, whereas others have relatively stringent substrate specificity, precluding PTMs at non-native sites.
Selective introduction of PTMs using non-canonical amino acids
So, is it possible to synthesize proteins with the desired post-translational modifications (PTMs) without relying on natural enzymes? By incorporating non‑natural amino acids (nnAAs or ncAAs) into a protein, one can selectively introduce the PTMs of interest at any desired site within the target protein.
Figure 1. Incorporation of non-canonical amino acids into proteins via genetic code expansion [1]
There are three main approaches to introducing site-specific PTMs by inserting nnAA:
① Directly incorporate the target-modified nnAA. Once the target protein is synthesized, it will possess the desired PTM.
② Incorporate nnAAs containing masked PTMs. After translation, the protecting groups can be removed by (photo)chemical methods, yielding proteins suitable for downstream applications.
③ Incorporate nnAA containing a chemical handle, which can be used to introduce the desired PTM via subsequent bioconjugation reactions. [1] 。
Figure 2. Introduction of PTMs by incorporating various non-canonical amino acids. [1]
Case of Introducing Phosphorylation Modifications Using Non-Natural Amino Acids
Phosphorylation, the reversible attachment of a phosphate group to a protein, is one of the most important post-translational modifications (PTMs) in nature and serves as a mechanism for diversifying protein function and molecular recognition. Javin et al. [2] employed a cell-free protein synthesis system (CFPS) to successfully express active human MEK1 kinase by introducing phosphorylated serine residues (Sep) at specific sites, thereby evaluating how site-specific phosphorylation—both mono‑ and di‑phosphorylated forms—contributes to MEK1 activity.
First, the researchers constructed a cell-free protein synthesis (CFPS) system using cell extracts derived from Escherichia coli. This E. coli strain is deficient in release factor 1 (RF1) and serine‑phosphate‑specific phosphatase (SerB), yet it expresses an orthogonal translation system for serine‑phosphate (Sep‑OTS) during growth. Consequently, the resulting CFPS system is enriched with the OTS components essential for phosphoprotein synthesis—namely, serine‑phosphate‑tRNA synthetase, tRNASep, and EF‑Sep.
Figure 3. Synthesis of phosphoproteins via CFPS-mediated Sep incorporation. [2]
Subsequently, the researchers used this CFPS system to express, within 20 hours, wild-type MEK1 with serine at positions S218 and S222 (MEK1-SS), MEK1 variants with single-site phosphorylation at either S218 or S222 (MEK1-SPS, MEK1-SSP), and a doubly phosphorylated MEK1 variant (MEK1-SPSP). After successfully expressing these different forms of MEK1, the researchers confirmed the production of full-length MEK1 and its various mutants by Western blot analysis; they further verified the presence of phospho‑Ser residues using Phos‑Tag gel shift assays, clearly demonstrating both single‑site and double‑site phosphorylation of MEK1. Finally, the researchers examined the enzymatic activity of the CFPS‑expressed mono‑phosphorylated and di‑phosphorylated MEK1 variants toward ERK2 in an in vitro kinase cascade assay.
Figure 4. In vitro synthesis of phosphorylated MEK1 variants [2]
The unique feature of this study in introducing phosphorylated MEK1 expression via the CFPS system is as follows: First, the CFPS system circumvents the need for transmembrane transport of the nnAA; in this work, the expression level of the MEK1 mutant was far higher than that achieved in cellular expression (0.001 mg/mL) [3]. Second, the CFPS system enables the expression of MEK1 without a fusion partner, thereby avoiding the confounding effects of soluble protein chaperones. Third, by introducing a Sep at a specific site, it is possible to generate a monophosphorylated form of the kinase without requiring alanine mutations to suppress phosphorylation at a second site.
The CFPS’s innate ability to incorporate non-natural amino acids
Cell-free protein synthesis (CFPS) is a biochemical reaction in which the molecular machinery required for intracellular protein synthesis—such as RNA polymerase, ribosomes, and transcription‑translation cofactors—is isolated and supplemented with nucleotides, amino acids, energy substrates, and a genetic template, enabling the direct in vitro production of proteins without the involvement of living cells.
CFPS possesses significant advantages in the field of non-natural amino acid incorporation:
(1) Cell-free systems lack a cellular membrane barrier, thereby circumventing the challenges associated with the transmembrane transport of nnAAs.
(2) Cell-free systems are not subject to the cytotoxic effects of nnAAs or of target non‑natural proteins.
(3) It helps eliminate endogenous competition for nnAA insertion, thereby enhancing the efficiency of nnAA incorporation.
References:
[1]NIU W, GUO J. Co-translational Installation of Posttranslational Modifications by Non-canonical Amino Acid Mutagenesis. ChemBioChem, 2023, 24(9).
[2]OZA J P, AERNI H R, PIRMAN N L, et al. Robust production of recombinant phosphoproteins using cell-free protein synthesis. Nature Communications, 2015, 6.
[3]Park, H.-S., et al. Expanding the genetic code of Escherichia coli with phosphoserine. Science, 2011, 333.
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