Incorporation of Unnatural Amino Acids

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The ability to incorporate unnatural amino acids (uAAs) into proteins has revolutionized modern enzyme engineering, enabling scientists to transcend natural biochemical boundaries. By introducing modified chemical functionalities, structural diversity, and unique physicochemical properties, unnatural amino acid incorporation provides a powerful platform for creating enzymes with enhanced or wholly novel catalytic capabilities. Creative Enzymes offers advanced, high-precision services for uAA incorporation across multiple systems, supported by decades of expertise in tRNA engineering, stop-codon reassignment, and codon expansion strategies. Our services are designed to facilitate structural exploration, mechanistic investigation, and functional enhancement of enzymes through seamless, reliable incorporation of noncanonical amino acids. Whether your goal is mechanistic probing, enhanced stability, unique catalytic activities, optical labeling, crosslinking capability, or therapeutic innovation, Creative Enzymes delivers the technical solutions and scientific insight needed to achieve exceptional results.

Introduction to Unnatural Amino Acid Incorporation

Enzyme engineering has traditionally relied on the 20 canonical amino acids that constitute the genetic code. While these natural residues offer a remarkable range of chemical properties, they cannot fully satisfy the increasingly sophisticated requirements of modern biotechnology, chemical biology, synthetic biology, and protein therapeutics. To overcome the limitations of natural amino acid side chains, researchers have developed technologies that allow site-specific incorporation of unnatural amino acids into proteins during translation. These methods expand the genetic code, introducing new chemical structures that provide unprecedented control over enzyme architecture and function.

Method to incorporate unnatural amino acids: amber codon suppression Figure 1. Incorporation of uAAs into proteins allows selective modifications. (Wals and Ovaa, 2014)

Purposes

Unnatural amino acid incorporation enables:

Novel catalytic functions through integration of chemically reactive moieties not found in nature.
Improved enzyme stability, including enhanced thermal or solvent tolerance.
Mechanistic insights, allowing incorporation of spectroscopic or photoactivatable probes.
Precise structural modification, enabling rigidification, crosslinking, or controlled conformational changes.
Biorthogonal reactivity, establishing selective chemical handles ideal for labeling or conjugation.

Creative Enzymes actively develops new uAAs and continuously enhances its incorporation methodologies. With decades of expertise in tRNA analogue design, stop codon editing, and both in vivo and in vitro translation systems, we offer comprehensive solutions that empower researchers to explore enzyme functionality beyond natural constraints. Our downstream services are fully customizable, ensuring seamless integration of uAA incorporation into broader enzyme engineering workflows.

What We Offer: Unnatural Amino Acid Incorporation Services

Creative Enzymes provides an extensive portfolio of services related to the design, optimization, and implementation of unnatural amino acid incorporation technologies. Our offerings include:

Service	Description	Price
Custom Design of Non-Canonical tRNA/Synthetase Systems	We develop tailored non-canonical tRNA and aminoacyl-tRNA synthetase systems that selectively recognize specific unnatural amino acids. This service enables precise and efficient incorporation of novel chemical functionalities into target proteins.	Inquiry
Engineering of Orthogonal tRNA/aaRS Pairs	Our team engineers highly selective orthogonal tRNA/aaRS pairs that operate independently of host translation machinery. These optimized systems support reliable site-specific introduction of a wide range of unnatural amino acids.
Codon Expansion and Genetic Code Reprogramming	We provide codon reassignment and genetic code reprogramming strategies to extend the natural coding capacity. This allows researchers to encode new amino acid chemistries directly into proteins.
Expression of Proteins with UAG or Quadruplet Codons	We specialize in expressing proteins that incorporate unnatural amino acids through UAG suppression or engineered quadruplet codons. Our optimized systems ensure high incorporation fidelity and robust protein yields.

Our service packages can be combined, expanded, or adapted according to your project needs. Whether you require a single uAA incorporation event or the construction of complex multi-site variants, Creative Enzymes provides the expertise and resources required to support all stages of development.

Service Workflow

Workflow of unnatural amino acid incorporation services

Service Features

Our uAA incorporation services cover the full spectrum of techniques and tools required for precise genetic code expansion.

Codon Expansion and Utilization

Creative Enzymes specializes in both traditional and advanced codon expansion methods:

Amber codon suppression (UAG)
Opal codon reassignment (UGA) for special use cases
Four-base codon systems for large-scale or multi-site incorporation
Sense codon reassignment for specialized applications requiring higher incorporation frequency

These strategies allow introduction of uAAs at specified locations with high fidelity.

tRNA and aaRS Engineering

We design engineered tRNA/aaRS pairs optimized for:

Broad substrate tolerance
Highly selective uAA activation
Compatibility with your expression system
Minimal interference with endogenous translation processes

Our team has decades of experience creating orthogonal translation systems compatible with both prokaryotic and eukaryotic machinery.

In Vivo Incorporation Platforms

Suitable for:

Structural modification
Protein functionality engineering
In vivo mechanistic studies
Preclinical evaluation of therapeutic proteins

Our bacterial and eukaryotic expression platforms support precise and efficient uAA incorporation with scalable yields.

In Vitro Incorporation Methods

Cell-free systems offer unmatched flexibility:

No membrane barriers
Controlled reaction environments
Ability to incorporate toxic or structurally challenging uAAs
Rapid reaction cycles ideal for high-throughput screening

Types of Unnatural Amino Acids Supported

We work routinely with a wide variety of uAAs including:

Photo-crosslinkable amino acids
Fluorescent and spectroscopic probes
Metal-binding residues
Keto, azide, and alkyne groups for biorthogonal chemistry
Halogenated, methylated, and aromatic analogues
Backbone-modified or sterically rigid residues
Redox-active or catalytic mimic amino acids

If you wish to develop a novel uAA, we can assist with synthetic design and translational compatibility assessment.

Scale and Deliverables

Depending on project needs, we provide:

Analytical-scale incorporation
Preparative-scale protein production
Library-scale incorporation for combinatorial design
Fully validated protein samples with accompanying documentation

Each project includes a complete report detailing methods, parameters, validation results, and recommendations for further development.

Contact Our Team

Why Choose Creative Enzymes

Decades of Professional Expertise

We possess extensive experience in tRNA analogue design, stop codon editing, and translation engineering.

Comprehensive Technical Capabilities

Methods covered include amber suppression, advanced four-base codon strategies, and fully orthogonal translation systems.

Cutting-Edge Customization

We give every project customized attention and tailor the workflow to meet your specific needs.

High Accuracy and Reliability

Our rigorous QC procedures ensure: High incorporation fidelity, minimal misincorporation, reliable expression yields, and accurate structural and functional validation.

Broad Compatibility With Diverse Expression Systems

We support both in vivo and in vitro platforms, enabling flexibility in project design.

Dedicated Scientific Support

Our team provides ongoing consultation at every stage—from concept evaluation and system selection to troubleshooting and downstream optimization.

Case Studies: Unnatural Amino Acid Incorporation

Case 1: Enhancing Enzyme Activity Through Unnatural Amino Acid Incorporation

Unnatural amino acid (UAA) incorporation has enabled precise, site-specific introduction of more than 30 novel amino acid structures into proteins, expanding the chemical possibilities far beyond the natural repertoire. In a recent study, UAAs were used to enhance the catalytic efficiency of the prodrug-activating enzyme nitroreductase. By sampling diverse structural and electrostatic features at a single active-site position, researchers identified a variant with over 30-fold improved activity compared with the native residue and more than 2.3-fold improvement over the best natural amino acid. This case highlights the power of UAAs to fine-tune active sites and create enzymes with superior or broadened substrate capabilities.

Improving nature's enzyme active site with genetically encoded unnatural amino acids Figure 2. Catalytic efficiency of modified NTR enzymes: (a) k_cat/K_M values for natural and unnatural NTRs with CB1954 and (b) k_cat/K_M values for natural and unnatural NTRs with LH7. (Jackson et al., 2006)

Case 2: Enhancing Phosphotriesterase Activity with Unnatural Amino Acids

Bacterial phosphotriesterases already exhibit near-maximal turnover rates for hydrolyzing the pesticide paraoxon. To explore whether unnatural amino acids (UAAs) could further enhance activity and probe peripheral active-site roles, tyrosine 309 was replaced with L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco), L-(7-methylcoumarin-4-yl)ethylglycine, and selected natural residues. Kinetic studies revealed that the deprotonated 7-hydroxyl group of Hco accelerates the rate-limiting product release via electrostatic repulsion, resulting in an 8–11-fold activity improvement. This single rationally designed UAA mutation surpasses what is achievable with extensive natural amino acid mutagenesis, illustrating how UAAs open new sequence and functional space for enzyme engineering and evolution.

Improving a natural enzyme activity through incorporation of unnatural amino acids Figure 3. Replace the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. (Ugwumba et al., 2011)

Common Questions About Unnatural Amino Acids Incorporation

Q: What types of unnatural amino acids can Creative Enzymes incorporate?

A: We can introduce a wide variety of uAAs, including photo-reactive, fluorescent, metal-binding, biorthogonal, halogenated, or backbone-modified amino acids. Custom development is also available.
Q: How many incorporation sites can be engineered simultaneously?

A: Single-site incorporation is most common, but multi-site incorporation is possible using mutually orthogonal suppression systems or specially designed codon reassignment strategies.
Q: Which expression systems are available?

A: We offer incorporation services in bacterial, yeast, mammalian, and cell-free systems. The optimal platform depends on your protein's complexity and the uAA's chemical characteristics.
Q: What validation methods do you use to confirm successful incorporation?

A: We use mass spectrometry, chromatographic analysis, functional assays, and optional structural analysis to confirm successful incorporation and protein quality.
Q: Can Creative Enzymes design novel unnatural amino acids?

A: Yes. We actively develop new uAAs and assist clients in creating structurally unique amino acids tailored to specific applications.
Q: How do I initiate a project?

A: Simply contact us with your target protein, incorporation site(s), and desired uAA. Our team will evaluate feasibility and provide recommendations, timelines, and pricing details.

References:

Jackson JC, Duffy SP, Hess KR, Mehl RA. Improving nature's enzyme active site with genetically encoded unnatural amino acids. J Am Chem Soc. 2006;128(34):11124-11127. doi:10.1021/ja061099y
Ugwumba IN, Ozawa K, Xu ZQ, et al. Improving a natural enzyme activity through incorporation of unnatural amino acids. J Am Chem Soc. 2011;133(2):326-333. doi:10.1021/ja106416g
Wals K, Ovaa H. Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins. Front Chem. 2014;2. doi:10.3389/fchem.2014.00015

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For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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