PUREfrex® represents a breakthrough in cell-free protein synthesis technology, offering a precisely engineered in vitro coupled transcription/translation system. Unlike traditional S30 translation extracts, PUREfrex® utilizes a completely reconstituted system derived from E. coli translation machinery.
PUREfrex®: Advanced Cell-Free Protein Synthesis System
Superior Protein Synthesis Through Precision Engineering
This innovative system combines highly purified components essential for protein synthesis:
- Purified E. coli ribosomes
- Specialized recombinant proteins
- Refined transcription factors
- Advanced translation elements
- Optimized aminoacylation components
- Enhanced energy regeneration systems
Key Advantages of PUREfrex® Technology:
- Unprecedented reaction control compared to extract-based systems
- Well-defined molecular composition for superior protein purification
- Enhanced reproducibility through standardized components
- Optimized yield through precise component ratios
Applications in Protein Research:
The defined nature of PUREfrex® makes it ideal for:
- Structural biology studies
- Protein engineering
- Synthetic biology applications
- Mechanistic investigations of protein synthesis
Applications of PUREfrex Cell-Free Protein Synthesis System
Protein Expression Capabilities
- Expression of prokaryotic and eukaryotic proteins with high efficiency
- Synthesis of complex membrane proteins and potentially cytotoxic protein products
- Production of proteins containing disulfide bonds with native conformations
- Generation of site-specifically labeled proteins for analytical applications
- Incorporation of non-canonical amino acids for protein modification and engineering
- Expression of truncated protein variants for structure-function analysis and epitope identification
Research Applications
- Verification and analysis of open reading frame (ORF) functionality
- Assessment of mutational effects on protein expression and ORF integrity
- Evaluation of amino acid substitutions on protein structure and function
- Implementation of ribosome display technology for protein evolution
- Investigation of fundamental translation mechanisms
- Advanced synthetic biology applications
- Protein folding mechanism studies
- Development of in vitro compartmentalization systems
Superior Purification Technology
The PUREfrex system employs advanced purification protocols to achieve exceptional component purity. This rigorous purification process effectively eliminates contaminating nucleases and proteases, ensuring:
- Preservation of DNA and RNA template integrity
- Maintenance of optimal protein functionality
- Maximum yield of full-length target proteins
PUREfrex 2.0 and 2.1 variants demonstrate significantly reduced RNase and β-galactosidase contamination levels. Additionally, lipopolysaccharide (LPS) concentrations have been reduced to approximately 0.1 Endotoxin Units (EU) per microliter of reaction mixture.
Streamlined Operational Protocol
The PUREfrex system features a simplified four-component format designed for operational efficiency:
- Solution I: Comprehensive non-protein component mixture
- Solution II: Purified protein component mixture
- Solution III: Isolated ribosome preparation
- Control Template: Verified DNA template for system validation
Template Requirements
Template preparation utilizes standard PCR methodology. Essential sequence elements include:
- T7 promoter sequence
- Ribosome binding site (Shine-Dalgarno sequence)
- Terminal stop codon
Protocol implementation requires only the combination of template DNA with Solutions I, II, and III, followed by incubation under specified conditions.
Enhanced Protein Component Design
The PUREfrex system represents an advancement over traditional PURE systems through its implementation of non-tagged protein components. This design feature offers several significant advantages:
Historical Context
Traditional PURE systems incorporated histidine-tagged (His-tagged) protein components to facilitate target protein purification. This approach enabled rapid isolation of the synthesized protein through negative selection using metal ion affinity chromatography (MIAC) following ribosome removal via centrifugation or filtration.
Advanced Component Design
PUREfrex employs untagged translation components, providing two key benefits:
-
Experimental Integrity
- Eliminates potential interference from tagged translation components
- Removes uncertainty regarding the impact of tags on protein synthesis efficiency
- Ensures more reliable experimental outcomes
-
Design Flexibility
- Enables unrestricted template design options
- Allows incorporation of any desired protein tag
- Supports production of completely untagged proteins
- Facilitates diverse experimental approaches and applications
This design enhancement allows researchers to optimize their experimental protocols according to specific research requirements without constraints imposed by system components.
PUREfrex® System Supplements: Advanced Functionalities and Applications
Overview of Supplement Functions
PUREfrex® supplements enhance the capabilities of base reaction mixtures through specialized components. These supplements are formulated for direct integration into standard PUREfrex® protocols, with concentrations and buffers optimized for immediate use. Consistent with the base system design, all protein supplements are produced without tags.
Molecular Chaperone Supplements
Purpose and Function
Molecular chaperones facilitate proper protein folding during synthesis. The base PUREfrex® systems do not include chaperones, making these supplements essential for proteins requiring assistance in achieving correct conformational states and solubility.
Available Chaperone Supplements
DnaK Mix
Components:
- Optimally-ratio balanced E. coli DnaK, DnaJ, and GrpE
- DnaK (Hsp70): Primary ATPase activity
- DnaJ: Enhances DnaK ATPase activity and binds hydrophobic protein regions
- GrpE: Facilitates DnaK ADP/ATP exchange
- Compatible with PUREfrex® series and DsbC Set
Figure. Effect of DnaK Mix on the activity of synthesized luciferase.
GroE Mix
Components:
- Ratio-optimized E. coli GroEL (Hsp60)
- GroES co-chaperone system
Figure. Effect of GroE Mix on the solubility of synthesized proteins.
(T: total reaction mixture, S: supernatant, P: precopitate fractions)
Redox Environment Supplements
Purpose
These supplements facilitate disulfide bond formation, crucial for secretory protein stability and folding. Efficacy depends on:
- Environmental redox conditions
- Presence of specialized isomerases
Available Redox Supplements
DsbC Set
Components:
- Purified E. coli DsbC (disulfide bond isomerase)
- Oxidized Glutathione (GSSG) for oxidizing conditions
PDI Set
Components:
- Oxidized Glutathione (GSSG)
- Human PDI (protein disulfide isomerase)
- Human Ero1α (maintains PDI oxidation state)
Translation Enhancement Supplements
EF-P Supplement
Function and Composition
- Contains modified Elongation Factor P (EF-P)
- Includes specialized dilution buffer
- Features post-translational β-lysyllysine modification at LYS34
Applications
- Enhances peptide bond formation between initial methionine and subsequent amino acids
- Facilitates translation of consecutive proline sequences
- Prevents ribosomal stalling
- Improves yield for proteins containing Pro-Pro-Pro and Pro-Pro-Gly sequences
Implementation Protocol
Workflow Optimization
- Template preparation and initial reaction
- Yield assessment
- Template sequence optimization if needed
- Supplement addition or reaction condition modification
- Verification of protein synthesis capability
Note: Each kit provides sufficient reagents for preliminary synthesis validation.
Ordering Information
PUREfrex Reaction Supplements
Depend on your protein’s property, we have supplements to add to PUREfrex® kit. Molecular chaperones, DnaK Mix or GroE Mix, are for improving folding and solubility. DsbC Set or PDI Set are for making disulfide bonds. Depending on each protein, adding those supplements to PUREfrex® kit helps effective translation and folding simultaneously.