CFPS for Non-natural Amino Acid Incorporation Service

Non-natural Amino Acid (nnAA) Incorporation via Cell-Free Protein Synthesis (CFPS) is a breakthrough technology that expands the genetic code, enabling the synthesis of proteins with novel functionalities beyond those achievable with the 20 canonical amino acids. By leveraging the open nature of CFPS systems, a genetically engineered orthogonal translation system is used to specifically incorporate an nnAA at a designated stop codon (TAG) within the target protein sequence.

CD Biosynsis offers a specialized CFPS for Non-natural Amino Acid Incorporation Service , providing the ideal platform for the rapid, site-specific production of novel functional proteins. Our service features high-yield CFPS extracts (E. coli, Wheat Germ, or Lysate systems) combined with our extensive library of over 50 orthogonal nnAAs . This approach eliminates issues of cell toxicity and transport limitations, accelerating the Design-Build-Test cycle for bioconjugates, novel enzymes, and structural probes . We guarantee site-specific incorporation and provide mandatory Mass Spectrometry (MS) verification for all custom nnAA-containing proteins.

Highlights

Core advantages of utilizing the open CFPS system for nnAA incorporation:

• Site-Specific Precision: Highly efficient incorporation of the nnAA at the precise location defined by the amber stop codon (TAG), ensuring homogenous protein products.
• Elimination of Cell Barriers: The open system allows direct addition of toxic, poorly permeable, or expensive nnAAs, bypassing cellular toxicity and transport limitations common in in vivo expression.
• Rapid Prototyping: Protein synthesis occurs within hours, dramatically accelerating the design-build-test cycle for protein engineering projects.
• High Yield for Difficult Proteins: Effective system for expressing proteins prone to aggregation or poor yield in cells, including cytotoxic and membrane proteins.

Applications

Expanding the genetic code for next-generation protein engineering and therapeutics:

Protein Bioconjugation (ADCs)

           

Incorporating functional handles (e.g., azide, alkyne) for highly selective Click Chemistry conjugation in Antibody-Drug Conjugates (ADCs) and protein labeling.

FRET & Structural Probes

Introducing fluorescent or spin labels at specific sites to monitor protein conformation, dynamics, and interaction in real time via FRET or EPR.

Enhanced Enzyme Catalysis

Introducing nnAAs with unique properties (e.g., enhanced acidity/basicity, metal binding) into enzyme active sites to improve catalytic efficiency or substrate specificity.

High-Throughput Screening

Rapidly producing large libraries of nnAA variants for directed evolution, aaRS engineering, and functional validation in a single day.

Key Features & CFPS Options

Our service provides flexible options and comprehensive technical support:

Integrated CFPS Systems

Access to optimized E. coli, Wheat Germ, and Rabbit Reticulocyte Lysate systems for optimal protein folding and compatibility with diverse downstream assays.

Extensive nnAA Library

Ready-to-use orthogonal systems for over 50 common nnAAs , including $p$-azido-L-phenylalanine, $p$-acetyl-L-phenylalanine, and various fluorophore-linked amino acids.

DNA Template Preparation

We perform the necessary site-directed mutagenesis (inserting the TAG codon) on your gene template and optimize the DNA template for maximal CFPS yield.

Mass Spectrometry QC

Mandatory MS analysis is performed to confirm full-length protein production and verify the exact mass shift corresponding to nnAA incorporation.

In-situ Functionalization

Optional post-translational modification or functionalization, such as Click Chemistry labeling of the nnAA handle, immediately after the CFPS reaction.

Mechanism & Workflow

Our integrated process ensures the high-fidelity synthesis of your custom modified protein:

• Gene Preparation: The gene of interest is engineered to contain the TAG stop codon at the desired incorporation site. The gene is cloned into an optimized CFPS expression vector.
• Orthogonal System Setup: The engineered aminoacyl-tRNA synthetase (aaRS) gene and the corresponding tRNA}_{CUA gene are added to the CFPS reaction mixture, often co-expressed from the same template.
• CFPS Reaction: The non-natural amino acid (nnAA) is added directly to the CFPS lysate, along with the necessary energy mix and the DNA template.
• Translation and Incorporation: When the ribosome encounters the TAG codon, the orthogonal tRNA}_{CUA (charged with the nnAA by the aaRS) is recruited, resulting in the site-specific incorporation of the nnAA into the nascent polypeptide chain.
• Purification and QC: The nnAA-containing protein is purified (typically via His-tag or Strep-tag) and analyzed by MS and SDS-PAGE to confirm incorporation efficiency and homogeneity.

We provide essential assurance for your expanded genetic code projects:

• Maximized Readthrough: Our E. coli CFPS systems are engineered to minimize the activity of Release Factor 1 (RF}1$) to maximize TAG codon readthrough and nnAA incorporation efficiency.
• Custom aaRS Engineering: For novel or highly challenging nnAAs, we offer directed evolution services to engineer a new, highly specific aaRS/tRNA pair.
• Purity and Homogeneity: We guarantee a high degree of site-specific incorporation, minimizing unwanted truncated or prematurely terminated products.
• Multi-Site Capability: Consultation and specialized systems for incorporating multiple different nnAAs into one protein using multiple distinct suppressor codons.