Sign In / Register          (0)
Incorporation of Unnatural Amino Acids
  1. Home
  2. Services
  3. Protein Production
  4. Protein Labeling
  5. Incorporation of Bioorthogonal Handles
  6. Incorporation of Unnatural Amino Acids

Incorporation of Unnatural Amino Acids

Background

Introduction

Unnatural amino acids (UAAs), engineered to augment the genetic code, empower proteins with novel functionalities like bioorthogonal reactivity, enhanced stability, and site-specific modifications. Emerging from early 2000s breakthroughs in genetic code expansion, UAA technology now drives innovations in antibody-drug conjugates (ADCs), enzyme engineering, and precision therapeutics. By integrating orthogonal tRNA systems and codon reassignment, UAAs enable tailored solutions for drug development and sustainable biomanufacturing, positioning them as pivotal tools in synthetic biology and personalized medicine.

Profacgen: Your Partner in Advanced UAA Solutions

Profacgen excels in UAA-based protein engineering, offering streamlined services from design to validation. With cost-effective workflows, rapid turnaround, and 24/7 support, Profacgen ensures scalable solutions for academia and industry.

Unlock your protein's potential-partner with Profacgen Now!

Strategies for UAA Incorporation

1. Metabolic Incorporation: Simplified UAA Integration Through Native Pathways

Metabolic incorporation replaces natural amino acids (e.g., methionine) with structural analogs like azidohomoalanine (AHA) by hijacking cellular biosynthesis pathways. Engineered auxotrophic hosts uptake supplemented UAAs, enabling seamless integration during protein synthesis without genetic code expansion. This approach prioritizes simplicity and scalability, ideal for large-scale production of labeled proteins or biomaterials. AHA's bioorthogonal azide group supports click chemistry for applications such as protein tracking or ADC development. Though limited to analogs resembling natural residues, metabolic engineering optimizes yield and specificity, making it cost-effective for industrial biocatalysis and therapeutic protein modification.

2. Site-Specific Incorporation: Precision Engineering via Orthogonal Systems

Site-specific UAA insertion uses engineered tRNA/aminoacyl-tRNA synthetase (aaRS) pairs and repurposed nonsense codons (e.g., amber STOP) to achieve residue-level control. Orthogonal systems, often derived from microbial sources, exclusively charge UAAs like p-azido-L-phenylalanine (pAzF) for targeted integration. This enables precise functionalization—such as photo-crosslinking or bioconjugation—at predefined sites, critical for developing ADCs, FRET biosensors, or stabilized therapeutics. Combining codon optimization, suppressor tRNA enhancements, and genome editing, platforms like Profacgen's ensure high fidelity, advancing synthetic biology and precision medicine.

Strategies for UAA Incorporation.Fig1. UAA incorporation utilizes tRNA/aaRS pair in response to an amber codon.

Applications of Unnatural Amino Acids

Enhancing Protein Functionality
  • Improving protein stability and reactivity
  • Modifying spectral properties for advanced imaging
Bioorthogonal Chemistry
  • Applications in click chemistry (Azides, Alkynes)
  • Staudinger ligation and other bioorthogonal reactions
Drug Discovery and Development
  • Incorporation of functional groups for drug targeting
  • Use in high-throughput screening and lead optimization
Synthetic Biology
  • Engineering proteins with novel functions
  • Creating synthetic pathways and metabolic circuits

Inquiry Now

Service Procedure

Sample Submission Requirements

To ensure the success of your UAA incorporation project, please follow these submission guidelines:

  1. Protein Sequence Information
    • Provide the full amino acid sequence of the target protein and specify the exact site(s) for UAA incorporation.
    • Indicate if codon optimization is needed for the expression system (e.g., E. coli, yeast, mammalian cells).
  2. UAA Specification
    • Specify the type of unnatural amino acid required (e.g., p-azido-L-phenylalanine, homopropargylglycine).
    • Indicate the desired bioorthogonal handle (e.g., azide, alkyne, ketone).
  3. Expression System
    • Specify the preferred expression system (e.g., E. coli, yeast, mammalian cells).
    • Note any specific vector requirements (e.g., plasmid backbone, promoter).
  4. Additional Information
    • Specify if any affinity tags (e.g., His-tag, GST-tag) are required.
    • Indicate purification requirements (e.g., Ni-NTA affinity chromatography).

Our UAA Incorporation Services

Experts at Profacgen provide the most up-to-date protein engineering technologies for incorporation of UAAs bearing bioorthogonal handles such as:

Profacgen offers comprehensive services for incorporating unnatural amino acids (UAAs) into proteins, tailored to meet your specific needs:

Customized Research Plans UAA Synthesis and Modification Protein Expression and Purification Characterization and Analysis Incorporation Strategies Application-Specific Solutions
  • Collaborate with you to design a customized plan for your UAA project.
  • Conduct feasibility assessments to determine the optimal approach.
  • Provide synthesis of UAAs with various bioorthogonal handles.
  • Introduce functional groups to enhance protein reactivity, stability, or imaging capabilities.
  • Utilize high-yield expression for recombinant protein production.
  • Employ advanced purification techniques to ensure high purity and yield.
  • Confirm UAA incorporation using high-resolution mass spectrometry (LC/MS).
  • Conduct functional assays to evaluate protein activity and stability.
  • Achieve site-specific UAA incorporation using orthogonal tRNA/aaRS pairs.
  • Offer metabolic incorporation of methionine analogs.
  • Support drug discovery by enhancing targeting and efficacy through UAA incorporation.
  • Enable synthetic biology by engineering proteins with novel functions.
  • Develop imaging probes with enhanced spectral properties.

Inquiry Now

Why Choose Profacgen?

  • Expertise and Experience: Over a decade of experience in protein engineering with a team of leading experts.
  • Advanced Technologies: State-of-the-art platforms for UAA synthesis, protein expression, and characterization.
  • Quality and Reliability: ISO-certified processes with stringent quality control for high success rates and reproducibility.
  • Customer Support: 24/7 customer service and technical support to assist you throughout your project.

Case Study

* NOTE: We prioritize confidentiality in our services to safeguard technology and intellectual property for enhanced future value and protection. The following case study has been shared with the client's consent.

Project: Custom Protein Expression with Unnatural Amino Acid (UAA) Incorporation

Goal

The project involves pilot protein expression in an E. coli system with the incorporation of an unnatural amino acid (UAA). The specific amino acid site in the protein sequence is replaced with an amber codon (UAG), and a modified tRNA synthetase/tRNA pair is used to incorporate the UAA in vivo. The process starts with gene synthesis, codon optimization, and vector construction, followed by protein expression and purification. The primary goal is to express a protein in the E. coli system with the incorporation of an unnatural amino acid (UAA).

Results

The project successfully utilized a tRNA/aaRS pair to incorporate the UAA in response to an amber codon. The engineered aminoacyl-tRNA synthetase (aaRS) specifically recognized its cognate tRNA without interfering with endogenous tRNAs. The full-length protein containing the UAA was isolated and detected using a C-terminal His tag. The protein mutant (carrying an amber codon at a specific site upstream of the C-terminal His tag) was successfully expressed in the presence of the engineered aaRS.

Result of protein expression.Fig2. Protein expression validation.

Lane M: Protein Marker; Lane 1: Wild protein

Lane 2: Protein mutant (contain amber codon at a specific site)

Lane 3: Protein mutant and engineered aaRS co expression.

Conclusions and Discussions

The project successfully synthesized a recombinant protein with a pAzF mutant gene and cloned it with a C-terminal His tag. The recombinant plasmid was co-expressed with the engineered aaRS in E. coli cells, and protein expression was successfully achieved.

Customer Testimonials

"Integrating unnatural amino acids via ProfacGen's platform revolutionized our antibody-drug conjugate (ADC) development. The site-specific incorporation improved payload precision in monoclonal antibodies, accelerating preclinical timelines. Their expertise in bioorthogonal chemistry saved us 3 months of method optimization. A must-try for targeted cancer therapy R&D teams!"

Dr. Emily Carter, Senior Research Lead | Biopharmaceutical Firm

"Exceptional PURE cell-free system support for engineered ribosomes! We achieved 92% incorporation efficiency of nitrotyrosine in artificial extracellular matrix proteins. Perfect balance between cost-effective solutions and high-yield expression systems. Critical for our tissue engineering projects requiring non-canonical amino acids."

Hiroshi Tanaka, CTO | Synthetic Biology Startup

"Game-changing tRNA synthetase engineering services for plant-based protein modification. Enabled stable pyrrolysine integration in drought-resistant crop enzymes. Their metabolic pathway optimization protocol outperformed two competitors we tested. Essential for sustainable agriculture innovation."

Dr. Priya Varma, Lab Director | Agricultural Biotech Co.

"Unmatched in amber stop codon suppression for viral vector vaccines. Achieved first-pass success with photocaged lysine in spike proteins for controlled immune activation. Their GMP-compatible platforms align perfectly with our mRNA-LNP formulation needs. Vital for next-gen vaccine platforms."

Michael O'Connor, Principal Scientist | Vaccine Developer

"Transformed our diagnostic assay development with clickable azidohomoalanine tags. Enabled rapid HRP/AP enzyme conjugation without compromising epitope binding. Their technical team's guidance on orthogonal translation systems was invaluable. Ideal for creating IVD kits requiring chemical biology precision."

Sofia Russo, Head of Proteomics | Diagnostic Tools Corp.

"Scaled up selenomethionine-incorporated industrial enzymes to 500L bioreactors using their modified CHO cell lines. Achieved 85% activity retention post-PEGylation – crucial for our detergent enzyme formulations. Their tech transfer documentation sets new industry benchmarks."

Dr. Liam Ng, Biomanufacturing Director | Industrial Enzymes Co.

FAQs

Q: What are the key advantages of incorporating unnatural amino acids (UAAs) in protein engineering?
A: Site-specific incorporation of unnatural amino acids enables precise post-translational modifications, unlocking functionalities like bioorthogonal chemistry handles (e.g., azides, alkynes), fluorescent tags, or photocaged residues. This enhances applications in antibody-drug conjugates (ADCs), mRNA-LNP vaccine design, and enzyme engineering. Unlike traditional methods, our engineered ribosomes and tRNA synthetase libraries ensure >90% incorporation efficiency while maintaining protein stability.
A: Absolutely. Our GMP-compatible CHO cell lines and cell-free protein synthesis systems allow scalable production of UAA-containing proteins (up to 500L bioreactors). For industrial enzymes or monoclonal antibody therapies, we optimize metabolic pathway engineering to minimize cost and maximize yield. Recent projects achieved 85% activity retention in PEGylated enzymes for detergent formulations.
A: While CRISPR modifies genetic sequences, UAA incorporation focuses on post-translational functionalization. Our amber stop codon suppression and orthogonal translation systems allow precise insertion of non-canonical residues (e.g., selenomethionine, nitrotyrosine) without altering native protein folding. This is ideal for targeted cancer therapies requiring controlled drug-payload attachment or diagnostic assays needing clickable tags.
A: Yes. We've successfully integrated pyrrolysine analogs into drought-resistant crop enzymes using plant-optimized tRNA synthetases. Our metabolic flux analysis ensures minimal disruption to native pathways, making UAAs viable for sustainable agriculture projects. Clients have reported 30% faster enzyme activation in saline soils compared to wild-type variants.
A: We employ LC-MS/MS verification and HPLC quantification to validate UAA insertion sites and purity. For critical applications like viral vector vaccines or IVD kits, we add bioactivity assays and epitope-binding tests. Our ISO-certified workflows guarantee batch-to-batch consistency, with documentation compliant with FDA/EMA guidelines for therapeutic proteins.

Resources

Bacterial Expression Fusion Protein Expression Stable Cell Line Construction for Protein Expression Protein Interaction Analysis Services Screening and Profiling Services Protein Labeling

References:

  1. Niu W, Guo J. Cellular Site-Specific Incorporation of Noncanonical Amino Acids in Synthetic Biology. Chem Rev. 2024;124(18):10577-10617.
  2. Singh-Blom A.; et al. Residue-specific incorporation of unnatural amino acids into proteins in vitro and in vivo. Methods Mol Biol. 2013;978:93-114.
  3. Cox VE, Gaucher EA. Molecular Evolution Directs Protein Translation Using Unnatural Amino Acids. Curr Protoc Chem Biol. 2015;7(4):223-228.
Online Inquiry

Fill out this form and one of our experts will respond to you within one business day.