CFPS Isotope Labeling for NMR/Structure Service

Stable isotope labeling of proteins is a prerequisite for advanced structural biology techniques, particularly Nuclear Magnetic Resonance (NMR) spectroscopy and certain mass spectrometry (MS) applications. The goal is to incorporate isotopes like Carbon-13 (^13C), Nitrogen-15 (^15N), and Deuterium (^2H or D) into the protein backbone or side chains to simplify and enhance the resolution of structural data.

CD Biosynsis offers a specialized CFPS Isotope Labeling Service that overcomes the limitations of traditional in vivo (cellular) methods. Our Cell-Free Protein Synthesis (CFPS) platform allows for the direct addition of highly concentrated, defined isotopic precursors (e.g.,99% ^13C-Glucose) into the reaction mixture, ensuring nearly 100% labeling efficiency with minimal loss of expensive materials. This method is ideal for producing difficult-to-express proteins (e.g., cytotoxic or membrane proteins) and for generating site-specific or segment-specific labeling for complex structural studies. We guarantee the highest purity and labeling fidelity required for high-resolution NMR analysis.

Highlights

Why CFPS is the superior method for isotope labeling compared to in vivo expression:

• Cost Efficiency: CFPS systems require significantly less isotopic media than cell culture, drastically reducing the cost of expensive precursors (^13C Glucose, D}_2O).
• High Yield for Difficult Proteins: Successfully labels toxic, aggregation-prone, or membrane proteins that are often difficult or impossible to express in standard E. coli culture for NMR study.
• Maximum Labeling Fidelity: The defined, open nature of the lysate ensures precise control over the amino acid pool, resulting in minimal scrambling and near 100% labeling efficiency .
• Rapid Labeling: The synthesis reaction is completed in a matter of hours, accelerating the overall timeline for structural data acquisition.

Applications

Isotope-labeled proteins are essential for advanced structural and functional analysis:

Protein Structure Determination (NMR)

           

Production of uniform single (^15N) or double (^15N, ^13C) labeled protein for backbone assignment and 3D structure calculation.

Protein Dynamics & Kinetics

Generating selective or fractional labeled protein for analyzing flexibility, conformational changes, and protein folding kinetics.

Protein-Ligand Interaction Mapping

Using uniformly or specifically labeled protein targets to map binding sites and calculate dissociation constants ($K_D$) using NMR titration.

Advanced MS and X-ray Crystallography

Producing deuterated protein for neutron scattering or isotope-labeled protein standards for precise MS quantification (SILAC).

Labeling Options & Advantages

Our CFPS system offers highly flexible and advanced labeling strategies:

Uniform Labeling (U-^15N or U-^15N, ^13C)

The standard labeling method using minimal quantities of ^15NH}_4Cl and/or ^13C-Glucose for comprehensive structural assignment.

Deuteration (U-^2H, ^13C, ^15N)

Using D}_2O as the solvent, this method replaces exchangeable protons with deuterium, essential for large protein ($>30 kDa) NMR studies.

Selective Labeling (Amino Acid Specific)

Only one or a few specific amino acid types are labeled (e.g., ^13C-Methionine), ideal for focusing on active sites or specific regions.

Segmental Labeling (Protein Splicing)

Advanced CFPS capability to label only a defined N or C-terminal segment of a protein via protein ligation (e.g., intein splicing).

Methyl Group Labeling (Highly Sensitive)

Specific labeling of methyl groups (e.g., Val, Leu, Ile, Met) in a deuterated background for high-sensitivity NMR of very large complexes.

Workflow

Our integrated CFPS labeling workflow ensures efficient, high-purity protein production:

• Project Design: Consultation on the optimal labeling strategy (uniform, selective, or segmental) based on the target protein size and desired NMR experiment.
• CFPS Template Preparation: Optimization of the target gene's DNA template for maximal expression in the chosen lysate system (typically E. coli).
• Labeling Reaction: The CFPS reaction is executed in a minimal volume using precisely calculated amounts of isotopic precursors (e.g., ^15N-ammonium sulfate) to ensure maximum incorporation.
• Protein Purification: The expressed, labeled protein is purified to NMR-grade (>95% purity) via methods like affinity chromatography and size exclusion chromatography.
• Quality Control (QC): Purity is confirmed by SDS-PAGE. Labeling fidelity is confirmed by Mass Spectrometry (MS) to verify the total mass shift resulting from the incorporation of stable isotopes.
• Delivery: Delivery includes the purified, lyophilized protein and a detailed COA containing MS and purity data.

We provide essential assurance for high-quality structural studies:

• Guaranteed NMR Grade Purity : Purification protocols are optimized to remove contaminants that interfere with NMR acquisition, such as detergents and metal ions.
• Expertise in Difficult Proteins: Routine success in labeling challenging targets like GPCRs, membrane proteins, and large protein complexes.
• Minimized Isotope Scrambling: The defined nature of the CFPS lysate greatly reduces scrambling (the exchange of isotopes between different amino acids), ensuring cleaner NMR spectra.
• Flexible Scale-Up: Seamless transition from small-scale labeling tests to large-scale production (milligram quantities) required for high-field NMR experiments.