Incorporating unnatural amino acids into proteins is a core strategy in modern protein engineering and chemical biology research. By introducing unnatural amino acids (UAAs) with specialized functions into protein molecules, researchers can achieve precise control over protein structure, activity, and function. This technology not only expands the chemical diversity of proteins but also opens new possibilities for drug development, functional protein design, industrial enzyme optimization, and molecular probe studies. By combining in vitro synthesis, chemical modification, and site-specific integration methods, researchers can precisely insert functional groups—such as photosensitive, fluorescent, or crosslinkable moieties—while maintaining the natural folding and biological activity of the protein, laying a solid foundation for analyzing complex biological systems and enabling innovative applications.
What are Unnatural Amino Acids?
Unnatural amino acids are amino acids that are not encoded by the natural genetic code. They can be obtained through chemical synthesis, semi-synthesis, or biosynthesis, and are typically derived from modifications of natural amino acid backbones or side-chain substitutions. UAAs may contain a variety of chemical functional groups, such as amides, azides, alkenes, aromatic groups, or fluorescent moieties, providing proteins with chemical reactivity or physical properties not available in natural amino acids. Based on their chemical structure and function, UAAs can be categorized as follows:
Structural-modifying UAAs: Alter side-chain length, rigidity, or chirality to stabilize protein structure or achieve unique folding patterns.
Functional group UAAs: Introduce reactive groups (e.g., azides, nitro, allyl) for subsequent chemical labeling or crosslinking.
Optically active or photoresponsive UAAs: Contain fluorescent or photosensitive groups for protein imaging, light-controlled function regulation, or photochemical crosslinking.
Chemically modified UAAs: Backbone or terminal modifications that provide enzymatic resistance, drug delivery capabilities, or enhanced targeting.
Key Strategies for Incorporating Unnatural Amino Acids into Proteins
Incorporating UAAs into proteins is a complex engineering task involving chemical synthesis, translation system modification, and precise control. Depending on the incorporation approach, strategies can be categorized into four types: in vitro protein synthesis, genetic code expansion, post-translational chemical modification, and peptide ligation techniques.
In Vitro Protein Synthesis: Cell-Free Systems for High Precision
Cell-free protein synthesis systems allow protein translation in acellular environments, including cell extract systems and PURE systems. This approach offers unique advantages for UAA incorporation:
Technical Principles
Mix mRNA, ribosomes, tRNAs, amino acids, and translation factors in vitro to directly synthesize proteins.
Provide UAAs directly in the system to replace specific natural amino acids for targeted protein modification.
Independent of cell membranes or metabolism, suitable for UAAs that are toxic or impermeable to cells.
Genetic Code Expansion: Site-Specific Incorporation in Living Cells
Genetic code expansion is the most widely used UAA incorporation strategy in living cells. Its core concept is to expand the genetic code so the cellular translation system can recognize UAAs.
Technical Principles
Rare or stop codon selection: The amber stop codon (UAG) is most commonly used due to its low natural occurrence, serving as a UAA insertion site.
Engineered tRNA/aaRS systems: Design specific tRNA/aaRS pairs so the aaRS recognizes only the target UAA and loads it onto the corresponding tRNA.
Directed insertion during translation: When the ribosome encounters the target codon (e.g., UAG), the specific tRNA inserts the UAA precisely at the designated site.
Implementation Systems
- coli: Early studies primarily use E. coli for producing small proteins and studying functionalization.
Yeast: Suitable for higher eukaryotic protein expression with glycosylation and folding capabilities.
Mammalian cells: Used for antibody therapeutics or complex protein functional optimization, achieving drug-grade protein modifications.
Post-Translational Chemical Modification: Functionalization After Translation
In addition to direct incorporation of unnatural amino acids during translation, chemical modifications can also be performed after protein synthesis to introduce specific functional groups.
Technical Principles
Utilize the side-chain functional groups of natural amino acids (e.g., Cys-SH, Lys-NH2, Tyr-OH) to selectively react with chemical reagents, thereby introducing properties of unnatural amino acids.
Common bioorthogonal chemistry methods include azide-alkyne cycloaddition (Click Chemistry), ketone/aldehyde–hydroxylamine reactions, and photosensitive crosslinking.
Modification sites can be precisely controlled without affecting the function of natural amino acids.
Chemical Synthesis Strategies: Peptide Ligation Techniques
For small to medium-sized proteins (typically fewer than 150 amino acids), fully chemical synthesis in vitro allows precise incorporation of UAAs with any chemical structure.
Solid-Phase Peptide Synthesis (SPPS)
Description: Uses automated instruments to sequentially couple amino acids from the C-terminus to the N-terminus on a solid resin.
Chemical Ligation
To synthesize larger proteins, segmental synthesis and ligation strategies are commonly used:
Native Chemical Ligation (NCL): Two chemically synthesized short peptides react in aqueous solution to form a longer peptide. One peptide carries a C-terminal α-thioester, and the other carries an N-terminal cysteine. After ligation, a natural peptide bond is formed at the junction.
Extended Ligation Techniques (e.g., β-thioester ligation): Modify NCL sites to allow ligation at non-cysteine positions, greatly increasing flexibility for synthesizing long proteins.
Applications: Chemical synthesis is the standard approach for obtaining highly pure, homogeneous UAA-modified proteins, particularly suitable for structural biology studies and drug development where moderate protein lengths and extremely high purity are required.
