Principles, System Types, and Experimental Application Design of Cell-Free Protein Synthesis Systems

A cell-free protein synthesis (CFPS) system is an in vitro reaction platform that uses cell lysates or reconstituted translation systems to synthesize target proteins from DNA, PCR products, or mRNA templates. This system operates independently of cell growth, transformation screening, and host toxicity constraints, making it suitable for toxic proteins, membrane proteins, difficult-to-express proteins, unnatural amino acid incorporation, rapid protein screening, synthetic biology circuit validation, and small-scale functional verification.

Keywords: cell-free protein synthesis; CFPS; in vitro translation; cell lysate; PURE system; membrane protein expression; unnatural amino acid; rapid protein screening

1 Basic Components of Cell-Free Protein Synthesis Systems

1.1 Reaction System Composition

(1) Genetic template

CFPS can use plasmid DNA, linear DNA, PCR products, or mRNA as templates. Plasmid DNA has relatively good stability and is suitable for routine expression and method optimization. PCR products do not require cloning and are suitable for high-throughput screening and rapid validation of mutants. mRNA templates bypass the transcription step and are suitable for analyzing translation efficiency and the influence of mRNA structure on protein yield. Linear DNA and PCR templates are susceptible to nuclease degradation, so terminal protection structures or nuclease inhibition strategies usually need to be optimized.

(2) Transcription and translation machinery

DNA-template-based CFPS requires RNA polymerase for transcription and relies on ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation factors, elongation factors, and termination factors for translation. Cell lysate systems usually retain these components and are relatively easy to operate. Reconstituted systems use purified components for reassembly, offering lower background and stronger controllability, but they are more costly and require more intensive component optimization.

(3) Substrates and energy system

Protein synthesis requires 20 amino acids, ATP, GTP, nucleotides, magnesium ions, potassium ions, buffer components, and an energy regeneration system. Common energy substrates include phosphoenolpyruvate, creatine phosphate, 3-phosphoglycerate, maltodextrin, and glucose-related systems. The energy system affects not only yield but also pH, inorganic phosphate accumulation, and effective magnesium ion concentration, thereby influencing translation duration.

(4) Auxiliary factors and reaction environment

Magnesium ions, potassium ions, disulfide bond-forming environments, molecular chaperones, detergents, liposomes, nanodiscs, and membrane-mimicking systems affect protein folding, solubility, and activity. When expressing soluble enzymes, membrane proteins, disulfide bond-containing proteins, or proteins containing unnatural amino acids, the same reaction composition should not be directly applied without adjustment.

Table 1 Core components of cell-free protein synthesis systems and their experimental roles

Component Type	Representative Components	Main Function	Experimental Focus
Genetic template	Plasmid DNA, PCR product, linear DNA, mRNA	Provides coding information for the target protein	Purity, concentration, promoter, UTR, degradation risk
Transcription system	T7 RNA polymerase, SP6 RNA polymerase, endogenous RNA polymerase	Generates mRNA from DNA	Promoter matching, transcription efficiency, mRNA stability
Translation system	Ribosomes, tRNAs, translation factors, aminoacyl-tRNA synthetases	Synthesizes polypeptide chains	Extract quality, translation factor activity, ionic conditions
Substrate system	Amino acids, nucleotides, energy substrates	Supports protein synthesis reaction	Substrate concentration, batch stability, energy regeneration efficiency
Folding assistance	Molecular chaperones, redox buffer, DsbC, PDI	Promotes correct folding and disulfide bond formation	Suitable for complex proteins or secretory proteins
Membrane-mimicking system	Detergents, liposomes, nanodiscs, microsomal membranes	Supports membrane protein insertion and stabilization	Membrane environment, protein activity, downstream purification strategy
Detection system	Fluorescent proteins, radioactive labeling, Western blot, activity assay	Determines expression level and function	Distinguishes total expression, soluble expression, and functional expression

1.2 Major System Types

(1) Escherichia coli lysate system

The E. coli lysate system has fast reaction kinetics, relatively high yield, and comparatively low cost. It is suitable for prokaryotic proteins, enzymes, screening-based expression, and high-throughput optimization. Its main limitations are the lack of complex eukaryotic post-translational modification capability and the need for additional system engineering or reaction condition optimization for complex disulfide bond proteins, glycoproteins, and some membrane proteins.

(2) Wheat germ system

The wheat germ system is a eukaryotic source system suitable for expressing some eukaryotic proteins, protein complexes, and target proteins that are unfavorable in prokaryotic systems. This system has relatively low background nuclease and protease activities and a comparatively mild protein folding environment. However, reaction rate and cost usually differ from E. coli systems, making it suitable for structural and functional studies and eukaryotic protein screening.

(3) Rabbit reticulocyte lysate system

The rabbit reticulocyte lysate system is suitable for mRNA template translation, eukaryotic protein expression, and some translation regulation studies. This system contains eukaryotic translation factors and a relatively complete translation environment, but it has higher requirements for template design, mRNA quality, and reaction background control.

(4) Insect cell and mammalian cell lysate systems

Insect cell, HeLa cell, and other mammalian-derived systems are closer to complex eukaryotic expression environments. They are suitable for some membrane proteins, multidomain proteins, post-translational modification-related studies, and preparation of drug target proteins. These systems are more costly, and yield, background, and batch consistency need to be carefully validated.

(5) PURE reconstituted system

The PURE system is reconstituted from purified ribosomes, translation factors, tRNAs, aminoacyl-tRNA synthetases, and energy components. It has defined composition, low background, and strong controllability, making it suitable for mechanistic studies, orthogonal translation systems, genetic code expansion, and synthetic biology circuit construction. Its main limitations are high cost and limited support for protein folding and complex modifications.

Table 2 Common CFPS system types and application selection

System Type	Main Advantages	Suitable Applications	Main Limitations
E. coli lysate system	Fast reaction, high yield, low cost	Enzymes, prokaryotic proteins, high-throughput screening	Lacks complex eukaryotic modifications
Wheat germ system	Eukaryotic source, relatively mild folding environment	Eukaryotic proteins, structural and functional screening	Cost and yield require separate evaluation
Rabbit reticulocyte lysate system	Suitable for mRNA translation and eukaryotic translation studies	Translation regulation, eukaryotic protein expression	Higher requirements for template and background control
Insect cell lysate system	More suitable for some complex eukaryotic proteins	Drug targets, membrane proteins, modification-related proteins	Higher system cost
Mammalian cell lysate system	Closer to mammalian translation environment	Human proteins, functional proteins, drug development	Yield and batch stability require validation
PURE reconstituted system	Defined composition, low background, strong controllability	Genetic code expansion, synthetic circuits, mechanistic studies	High cost, limited folding assistance

2 Key Parameters in CFPS Experimental Design

2.1 Template Design and Template Quality

(1) Promoter and regulatory elements

DNA templates should match the RNA polymerase used in the reaction system. The T7 promoter is commonly used in E. coli lysate and PURE systems. The template should also include a properly designed ribosome binding site, start codon, stop codon, and 5′/3′ untranslated regions. Eukaryotic systems depend more strongly on mRNA cap structure, poly(A) tail, or UTR designs suitable for translation initiation.

(2) Template format

Plasmid DNA generally has better stability and is suitable for standard expression. PCR templates are suitable for rapid screening of mutants, domain boundaries, and tag positions. mRNA templates are suitable for directly evaluating translation efficiency. Linear DNA and PCR templates may be degraded by nucleases after entering the lysate; if necessary, protective sequences, nuclease inhibitors, or systems better suited for linear templates can be used.

(3) Template purity

Salt, ethanol, phenol, guanidine salts, EDTA, and protein contamination may inhibit transcription or translation reactions. After template preparation, concentration, integrity, and purity should be confirmed. PCR products in particular require removal of residual primers, dNTPs, polymerase, and buffer salts.

2.2 Ionic Conditions and Energy Regeneration

(1) Magnesium ions

Magnesium ions affect ribosome structure, tRNA binding, ATP/GTP utilization, and nucleic acid stability, and are among the most sensitive parameters in CFPS. Insufficient magnesium ions reduce translation efficiency, whereas excessive magnesium may cause nonspecific precipitation or translation inhibition. Magnesium concentration should be re-optimized for different templates, energy substrates, and lysate batches.

(2) Potassium ions

Potassium ions affect ribosome activity, protein folding, and the overall translation environment. E. coli systems are usually sensitive to potassium salt concentration, and eukaryotic systems also require ionic strength suitable for the translation machinery. Potassium optimization should be considered together with magnesium rather than adjusted independently.

(3) Energy substrates

The energy regeneration system determines reaction duration and yield. Phosphoenolpyruvate can supply energy rapidly, but byproducts and inorganic phosphate accumulation may affect the reaction. 3-Phosphoglycerate, creatine phosphate, maltodextrin, or glucose-related systems can be used to extend reaction duration or reduce cost. When selecting an energy system, yield, pH stability, cost, and target protein activity should be considered together.

2.3 Reaction Mode and Detection Strategy

(1) Batch reaction

Batch reactions are simple to operate and are suitable for expression condition screening, template comparison, tag validation, and high-throughput reactions. Their limitations are rapid substrate depletion, product inhibition, and byproduct accumulation, so reaction duration is usually limited.

(2) Continuous exchange reaction

Continuous exchange systems use a semipermeable membrane or compartmentalized structure to continuously replenish substrates and remove small-molecule byproducts, extending reaction time and increasing yield. This mode is suitable for yield scale-up, structural protein preparation, and protein synthesis requiring longer reaction times.

(3) Continuous-flow reaction

Continuous-flow systems enable substrate feeding, reaction volume scale-up, and more stable system control. They are suitable for process research and automated platform development. Design priorities include reactor structure, flow rate, template stability, and product collection efficiency.

(4) Detection strategy

CFPS results should distinguish “total expression,” “soluble expression,” and “functional product.” Fluorescent proteins are suitable for rapid reporting of expression capacity. SDS-PAGE and Western blot are suitable for detecting molecular weight and expression bands. Enzyme activity, binding assays, ligand response, or cell-based functional assays are used to confirm whether the protein has the intended function.

Table 3 Key CFPS parameters and optimization directions

Parameter	Affected Step	Common Problem	Optimization Direction
Template concentration	Transcription and translation initiation	Insufficient expression when too low; reaction inhibition when too high	Establish a template concentration gradient
Promoter/RBS/UTR	mRNA generation and translation initiation	mRNA is present but protein yield is low	Optimize promoter, RBS, or UTR structure
Magnesium ions	Ribosome and nucleic acid stability	Low yield, precipitation, or high background	Perform magnesium gradient optimization for each system
Potassium ions	Translation efficiency and protein folding	Fluctuating translation efficiency	Optimize together with magnesium ions
Energy system	Reaction duration	Premature reaction termination, pH drift	Replace energy substrate or buffer system
Reaction temperature	Translation rate and folding	Rapid expression but poor solubility	Lower temperature or extend reaction time
Redox environment	Disulfide bond formation	Inactive or mismatched protein	Add redox buffer and folding enzymes
Membrane-mimicking system	Membrane protein insertion	Aggregation or loss of function	Add liposomes, nanodiscs, or microsomal membranes

3 Applications of CFPS in Difficult-to-Express and Functional Protein Preparation

3.1 Toxic Protein Expression

(1) Avoiding host toxicity

When toxic proteins are expressed in cells, they may inhibit host growth, disrupt metabolism, damage cell membranes, or destabilize expression strains. CFPS does not rely on live cell proliferation and can directly synthesize target proteins in vitro, reducing host selection pressure during cloning and induced expression.

(2) Expression process control

When expressing toxic proteins, reaction volume, template concentration, reaction time, and product exposure risk should be controlled. If the protein has nuclease, protease, or membrane-disrupting activity, inactive mutants, no-template controls, and post-reaction inactivation steps should be included to avoid effects on system components and operators.

(3) Result verification

Successful toxic protein expression should not be judged only by an expression band. Purification results, activity assays, substrate specificity, and stability evaluation should be combined to distinguish full-length expression, degradation products, and nonspecific aggregation.

3.2 Membrane Protein Expression

(1) Requirement for membrane environment

Membrane proteins require a hydrophobic environment to maintain their conformation. Direct expression in aqueous CFPS systems can easily lead to aggregation or inactivation. Adding detergents, liposomes, nanodiscs, microsomal membranes, or artificial membrane systems can improve membrane protein insertion, folding, and functional detection.

(2) System selection

E. coli lysates are suitable for some prokaryotic membrane proteins and rapid screening. Wheat germ, insect cell, or mammalian systems are more suitable for some eukaryotic membrane proteins and complex drug targets. If the target protein requires glycosylation, disulfide bonds, or membrane localization-related modifications, systems containing microsomal membranes or eukaryotic-derived systems should be prioritized.

(3) Functional evaluation

After membrane protein expression, ligand binding, transport activity, receptor signaling, antibody recognition, or structural integrity should be examined. An SDS-PAGE band alone cannot prove that the membrane protein is correctly folded or inserted into the membrane environment.

3.3 Disulfide Bond-Containing and Complex Folding Proteins

(1) Redox conditions

Disulfide bond-containing proteins require an appropriate redox environment. Reducing components in cell lysates may hinder disulfide bond formation. This can be improved by adjusting the glutathione redox buffer system, adding disulfide bond isomerases, or pretreating lysates to reduce the reducing background.

(2) Folding auxiliary factors

DsbC, PDI, molecular chaperones, prolyl isomerases, and other auxiliary factors can improve folding efficiency of complex proteins. Therapeutic proteins, antibody fragments, cytokines, and multidisulfide proteins usually require simultaneous evaluation of expression level and activity.

(3) Activity detection

Functional assays should be prioritized for evaluating complex folded proteins. Catalytic activity can be measured for enzymes; ligand binding or antibody recognition can be measured for receptors or binding proteins; cell function or target response can be measured for therapeutic proteins.

4 Applications of CFPS in Synthetic Biology and R&D Platforms

4.1 Unnatural Amino Acids and Genetic Code Expansion

(1) Orthogonal translation system

CFPS can incorporate unnatural amino acids site-specifically into target proteins by adding an orthogonal tRNA/aminoacyl-tRNA synthetase pair. The composition of the in vitro system is controllable, which facilitates adjustment of unnatural amino acid concentration, stop codon suppression efficiency, and competitive translation conditions.

(2) Special protein design

Unnatural amino acids can be used to introduce fluorescent groups, crosslinking groups, click chemistry handles, photoreactive groups, or stabilizing structures. This strategy is suitable for protein labeling, structural analysis, drug conjugation, and functional regulation studies.

(3) Interpretation points

Unnatural amino acid incorporation experiments should confirm the proportion of full-length protein, misincorporation rate, truncated products, and modification efficiency at the target site. Mass spectrometry verification is more suitable than fluorescence or band detection alone for confirming site-specific incorporation.

4.2 High-Throughput Protein Screening

(1) Rapid template construction

PCR products can be directly used in CFPS reactions, making them suitable for domain boundary screening, mutant library screening, tag position comparison, and early-stage protein engineering validation. This mode reduces cloning time and accelerates candidate protein screening.

(2) Microplate reactions

CFPS can be carried out in small-volume reactions in microplates and is suitable for throughput testing of expression level, solubility, enzyme activity, fluorescence signal, or binding ability. After reaction miniaturization, evaporation, edge effects, and pipetting error should be re-evaluated.

(3) Data screening

High-throughput results should include positive template, negative template, no-template, and system background controls. Candidates with high expression but low function may have folding problems, while candidates with low expression but high activity may be more suitable for subsequent scale-up optimization.

4.3 Gene Circuit and Biosensor Validation

(1) Gene circuit prototyping

CFPS can be used to rapidly validate promoters, RBSs, riboswitches, transcription factors, and feedback loops in vitro. Because the system does not include cell growth or metabolic adaptation processes, it is suitable for analyzing the response logic of the circuit itself.

(2) Biosensors

In vitro biosensors can be constructed by combining transcription/translation systems with small-molecule-responsive elements. Such systems can be used in environmental detection, metabolite screening, pathogen-related signal detection, and field-deployable rapid testing platforms.

(3) Boundary conditions

In vitro circuit results are not necessarily identical to intracellular results. After transfer into cells, plasmid copy number, resource competition, metabolic state, protein degradation, and cell growth can alter circuit behavior. Therefore, CFPS is more suitable as a platform for prototype validation and parameter screening.

Table 4 CFPS application scenarios and experimental evaluation indicators

Application Direction	Main Goal	Recommended System	Key Evaluation Indicators
Toxic protein expression	Avoid host death and expression inhibition	E. coli lysate, PURE system	Expression level, integrity, activity, safety handling
Membrane protein preparation	Improve membrane protein expression and folding	Eukaryotic lysate, system with membrane mimics	Soluble/membrane-bound ratio, ligand binding, functional activity
Therapeutic protein validation	Rapidly obtain functional protein	Eukaryotic system or optimized lysate system	Folding state, disulfide bonds, cell function
Unnatural amino acid incorporation	Construct site-specifically modified proteins	PURE system, E. coli lysate	Full-length product, site incorporation rate, mass spectrometry results
High-throughput mutant screening	Rapidly compare candidate variants	PCR template + microplate CFPS	Expression level, solubility, enzyme activity, or binding ability
Gene circuit validation	Test regulatory elements and response logic	PURE system, lysate system	Fluorescence output, dynamic range, background signal
In vitro biosensor	Detect small-molecule or nucleic acid signals	Lyophilized CFPS or portable system	Sensitivity, specificity, stability

5 CFPS Result Interpretation and Common Problems

5.1 Low Expression Level

(1) Template problems

Insufficient template concentration, promoter mismatch, unreasonable RBS or UTR design, and excessive PCR residual inhibitors can all lead to low expression. The source of the problem can be identified by changing template format, optimizing the 5′ sequence, improving template purity, and including a positive template.

(2) Insufficient system activity

Cell lysate batch differences, excessive freeze-thaw cycles, decreased ribosome activity, or imbalance in the energy system can affect expression. A standard reporter protein should be used as a system quality control to avoid misinterpreting system inactivation as difficult expression of the target protein.

(3) Mismatched reaction conditions

Mismatched magnesium ions, potassium ions, temperature, reaction time, or energy substrate can reduce yield. Optimization should begin with a small matrix rather than changing only one parameter at a time and directly concluding that the system has failed.

5.2 Insoluble or Inactive Protein

(1) Insufficient folding

After expression, the target protein may form aggregates or misfolded products. Folding can be improved by lowering reaction temperature, extending reaction time, adding molecular chaperones, optimizing the redox environment, or switching to a eukaryotic system.

(2) Missing modifications

Glycosylation, phosphorylation, disulfide bonds, lipid modification, or membrane insertion can be essential for the function of some proteins. When these modifications are absent, the protein may remain inactive even if expression is high. Such targets should use more suitable eukaryotic lysates, microsomal membrane systems, or downstream in vitro modification strategies.

(3) Mismatched detection method

Some proteins are sensitive to tag position, buffer, cofactors, or substrate conditions. If the activity assay is negative, incompatibility of the assay system, insufficient cofactors, or tag interference should be excluded.

5.3 High Background or Poor Reproducibility

(1) Endogenous background

Lysates may contain endogenous proteins, nucleic acids, and metabolite background. When detecting low-expression proteins or weakly active proteins, no-template controls, blank template controls, and unrelated template controls should be included.

(2) Reaction system fluctuation

Microvolume reactions are susceptible to pipetting error, evaporation, and edge effects. In high-throughput experiments, replicate wells, randomized layout, and intra-plate standards should be used to control batch differences.

(3) Product degradation

Protease activity, nuclease activity, or intrinsic instability of the target protein can cause product degradation. Protease inhibitors can be added, reaction time optimized, temperature lowered, or lysate source changed.

Table 5 Common CFPS problems and optimization directions

Problem	Possible Cause	Optimization Direction
No expression or weak expression	Template degradation, promoter mismatch, system inactivation	Include a positive template; optimize template quality and promoter design
mRNA present but protein low	Unsuitable RBS/UTR, weak translation initiation	Optimize translation initiation elements and 5′ structure
High total expression but low solubility	Insufficient folding, reaction too fast, temperature too high	Lower temperature, add molecular chaperones, or change system
Disulfide bond-containing protein inactive	Inappropriate redox environment	Add GSSG/GSH, DsbC, or PDI
Membrane protein aggregation	Lack of membrane environment	Add liposomes, nanodiscs, detergents, or microsomal membranes
Poor high-throughput reproducibility	Pipetting error, evaporation, plate position effect	Increase replicate wells, seal plates, and include intra-plate standard controls
High background signal	Endogenous lysate background or detection interference	Include no-template and no-substrate controls

6 Related Product and Material Selection

Table 6 Products and materials related to cell-free protein synthesis systems

Application Module	Cat. No.	Product Name	Grade/Specification	Application Positioning
Core reaction system	C1522814	Cell-Free Protein Synthesis Kit	BioReagent；for protein analysis	Used for routine CFPS reactions, rapid target protein expression, and small-scale protein analysis
Core reaction system	C1522812	Cell-Free Protein Synthesis Kit (Lyophilized)	BioReagent；for protein analysis	Used for lyophilized CFPS systems, suitable for storage stability, portable reactions, and batch experimental design
Disulfide bond protein expression	C1522808	Cell-Free Protein Synthesis Kit Max	BioReagent；for protein analysis	Used for disulfide bond-containing proteins, secretory proteins, antibody fragments, and complex folded protein expression
Solubility-enhanced expression	C1522810	Cell-Free Protein Synthesis Kit Pro	BioReagent；for protein analysis	Used for aggregation-prone proteins, low-solubility proteins, and targets requiring improved soluble expression ratio
Disulfide bond protein expression	C1522801	Cell-Free Protein Synthesis Kit Max (Lyophilized)	BioReagent；for protein analysis	Used for lyophilized disulfide bond-enhanced CFPS systems, suitable for disulfide bond-containing protein expression and stabilized reaction preparation
Solubility-enhanced expression	C1522809	Cell-Free Protein Synthesis Kit Pro (Lyophilized)	BioReagent；for protein analysis	Used for lyophilized solubility-enhanced CFPS systems, suitable for difficult-to-solubilize proteins, screening-based expression, and portable protein synthesis experiments
Template preparation	D745686	EnzymoPure™ DNA Polymerase	EnzymoPure™, free of DNA endonuclease and exonuclease, phosphatase, and RNase.	Used to amplify CFPS linear DNA templates, expression cassettes, or screening PCR products
Template preparation	H745689	EnzymoPure™ High-Fidelity DNA Polymerase	EnzymoPure™, This product is free from RNase, phosphatase, and DNA endonuclease.	Used for high-fidelity amplification of target protein coding sequences and reducing template mutation risk
Template preparation	E745687	EnzymoPure™Extra-long DNA Polymerase	EnzymoPure™, free of DNA endonuclease and exonuclease, phosphatase, and RNase.	Used for amplification of long expression templates, composite regulatory elements, or long protein coding sequences
Template preparation	H745688	EnzymoPure™ HF DNA Polymerase (Blood-resistant)	EnzymoPure™, free of DNA endonuclease and exonuclease, phosphatase, and RNase.	Used for template amplification from complex sample sources or PCR systems with high inhibitor background
Nucleotide substrate	U665648	UltraPure dNTP Mix(10 mM each)	5 ml	Used for PCR template amplification and supports linear DNA template and mutant template preparation
Nucleotide substrate	U759856	UltraPure dATP (100mM)	BioReagent, DNase, RNase free, PCR Reagent, endotoxin tested, UltraBio™, Suitable for molecular biology, sterile, for DNA and RNA applications, ≥99%, 100mM	Used for PCR template amplification, in vitro DNA synthesis, or individually prepared dNTP systems
Nucleotide substrate	U759854	UltraPure dCTP (100mM)	BioReagent, DNase, RNase free, PCR Reagent, endotoxin tested, UltraBio™, Suitable for molecular biology, sterile, for DNA and RNA applications, ≥99%, 100mM	Used for PCR template amplification, in vitro DNA synthesis, or individually prepared dNTP systems
Nucleotide substrate	U759851	UltraPure dGTP (100mM)	BioReagent, DNase, RNase free, PCR Reagent, endotoxin tested, UltraBio™, Suitable for molecular biology, sterile, for DNA and RNA applications, ≥99%, 100mM	Used for PCR template amplification, in vitro DNA synthesis, or individually prepared dNTP systems
Nucleotide substrate	U759852	UltraPure dTTP (100mM)	BioReagent, DNase, RNase free, PCR Reagent, endotoxin tested, UltraBio™, Suitable for molecular biology, sterile, for DNA and RNA applications, ≥99%, 100mM	Used for PCR template amplification, in vitro DNA synthesis, or individually prepared dNTP systems
In vitro transcription	T292917	T7 RNA Polymerase	BioReagent,endotoxin tested,Suitable for molecular biology,EnzymoPure™,RNase free,Recombinant,≥98.0%,50 U/μL	Used for T7 promoter-driven in vitro transcription and coupled transcription-translation CFPS reactions
In vitro transcription	T1372515	T7 RNA Polymerase	ActiBioPure™, Carrier Free, Bioactive, EnzymoPure™, 1KU/μL	Used for high-concentration transcription systems, mRNA template preparation, and high-throughput CFPS template validation
RNA protection	R489312	RNase Inhibitor	pharmaceutical grade, PharmPure™, ≥95%, 1000U/μl	Used to protect mRNA templates and transcription products, reducing decreased translation efficiency caused by RNase contamination
RNA protection	R489315	RNase Inhibitor	pharmaceutical grade, PharmPure™, ≥95%, 40U/μl	Used in mRNA-template CFPS, in vitro transcription, and RNA sample handling for RNase inhibition
RNA protection	R350859	RNase Inhibitor (Murine, 40U/μL)	Recombinant, BioReagent, DNase, RNase free, Suitable for molecular biology, for DNA and RNA applications, ≥95%(SDS-PAGE), 40U/μl	Used for RNA-sensitive CFPS systems and mRNA template stability control
RNA protection	R292575	RNase Inhibitor（RNasin）	40U/μl	Used for RNA protection in in vitro transcription, mRNA template preparation, and translation reactions
RNA-grade solvent	R665526	RNase-Free Water	100 ml	Used to prepare CFPS reaction components, RNA templates, nucleotides, and buffers
Reaction buffer and dilution	P743267	PBS, DNase&RNase Free	sterile-filtered, BioReagent, DNase, RNase free, ready-to-use, for cell culture, 1×	Used for nucleic acid or protein sample dilution, pre- and post-reaction processing, and low-nuclease-background experiments
Reaction environment regulation	P748949	PEG8000 (50%, RNase free)	BioReagent, DNase, RNase free, Protease Free, ≥99%, 50%	Can be used for molecular crowding environments, RNA-related experiments, or CFPS condition optimization
Decontamination treatment	R666126	RNase and DNA Remover	250ml	Used to remove RNase/DNA contamination from benches, tools, and reaction areas, reducing CFPS background interference
Transcription substrate/energy substrate	A100880	Adenosine 5ʹ-Triphosphate, Disodium Salt (ATP)	≥98%	Used in transcription, translation, and energy cycling systems; a core nucleotide component of CFPS
Transcription substrate/energy substrate	A427208	Adenosine 5′-triphosphate disodium salt (ATP)	10mM in Water	Used for small-volume reactions, system optimization, and directly added ATP supplementation
Transcription substrate	G452636	GTP Trisodium salt Solution	pharmaceutical grade, PharmPure™, ≥99%, 100mM	Used for mRNA transcription, translation elongation, and CFPS nucleotide substrate supplementation
Transcription substrate	G427293	GTP Trisodium salt Solution	pharmaceutical grade, PharmPure™, ≥99%, 10mM	Used for small-volume CFPS reactions, nucleotide supplementation, and transcription system optimization
Transcription substrate	G122989	Guanosine-5'-triphosphate disodium salt（5′-GTP-Na2）	≥90%	Used in in vitro transcription, translation reactions, and GTP-related substrate systems
Transcription substrate	C427289	CTP Trisodium salt Solution	pharmaceutical grade, PharmPure™, ≥99%, 100mM	Used for RNA synthesis reactions and coupled transcription-translation systems
Transcription substrate	U427287	UTP Trisodium salt Solution	pharmaceutical grade, PharmPure™, ≥99%, 100mM	Used for RNA synthesis reactions and CFPS transcription substrate supplementation
Transcription substrate	U406318	Uridine 5′-triphosphate trisodium salt hydrate（UTP Na3）	Moligand™, ≥95%	Used for in vitro transcription reactions and mRNA template preparation
Modified RNA substrate	N464730	N1-Me-Pseudo UTP Trisodium solution	pharmaceutical grade, PharmPure™, ≥99%, 100mM	Used for modified RNA template preparation and experiments related to mRNA stability and translation efficiency
Modified RNA substrate	P463387	Pseudo UTP Trisodium solution	pharmaceutical grade, PharmPure™, ≥99%, 100mM	Used for pseudouridine-modified RNA preparation and mRNA template optimization
Energy regeneration	P423930	Phospho(enol)pyruvic acid monopotassium salt	10mM in Water	Used in PEP-based energy supply systems, supporting ATP regeneration and short-duration efficient protein synthesis
Energy regeneration	P119478	Phospho(enol)pyruvic acid monopotassium salt	≥97%	Used for CFPS energy regeneration system construction and reaction duration optimization
Energy regeneration	L1491909	Creatine Phosphokinase	EnzymoPure™, Native, ≥300 U/mg protein	Used as the ATP regeneration enzyme component in creatine phosphate energy systems
Energy regeneration	L1491908	Creatine Phosphokinase	EnzymoPure™, Native, >30 U/mg protein	Used for method validation in creatine phosphate/creatine kinase energy regeneration systems
Labeled amino acid substrate	C468508	Cell Free Amino Acid Mixture-¹³C, ¹⁵N, D	≥97 atom% D,≥98 atom% 13C,≥98 atom% 15N, 2-100mM in water	Used for isotope-labeled protein synthesis in CFPS, structural studies, and mass spectrometry analysis
Labeled amino acid substrate	C471877	Cell Free Amino Acid Mixture-¹⁵N	≥98 atom% 15N, 5-100mM in water	Used for ¹⁵N-labeled protein expression, NMR, or proteomics analysis
RNA/nucleic acid electrophoresis	B1518790	BPTE Running B uffer (10×, RNase Free)	BioReagent,Suitable for molecular biology,RNase free,for NA electrophoresis,10×	Used for quality analysis of mRNA templates, in vitro transcription products, or nucleic acid templates
RNA/nucleic acid electrophoresis	T1518783	TBE Buffer (5×, RNase Free)	BioReagent,Suitable for molecular biology,RNase free,for NA electrophoresis,5×	Used for electrophoretic detection of DNA templates, PCR products, and RNA samples
RNA/nucleic acid electrophoresis	T1518779	TAE Buffer (50×, RNase Free)	BioReagent,Suitable for molecular biology,for NA electrophoresis,RNase free,50×	Used for CFPS template quality control and nucleic acid integrity detection
RNA hybridization/nucleic acid analysis	S1518774	SSC Buffer (20×, pH 7.0, RNase Free)	BioReagent,Suitable for molecular biology,RNase free,sterile,for DNA and RNA applications,20×	Used as a low-RNase-background buffer system in RNA or DNA analysis workflows
Nucleic acid analysis buffer	S743320	SSPE	20X, pH7.4	Used for nucleic acid analysis, hybridization, or low-degradation-background sample processing

7 Frequently Asked Questions

7.1 Is a cell-free protein synthesis system suitable for replacing all cell-based expression systems?

No. CFPS is suitable for rapid expression, difficult-to-express protein screening, toxic proteins, membrane proteins, unnatural amino acid incorporation, and synthetic biology prototype validation. If the goal is low-cost large-scale production, complex post-translational modification, or stable secretory expression, the cost and functional advantages of cell-based expression systems and CFPS should still be compared according to protein type.

7.2 How should DNA templates and mRNA templates be selected?

DNA templates are suitable for coupled transcription-translation reactions, are easy to handle, and are commonly used in E. coli lysate and PURE systems. mRNA templates are suitable for directly studying translation efficiency and eukaryotic translation regulation, but they are more sensitive to RNA integrity and RNase contamination. PCR templates can be prioritized for high-throughput screening, while plasmid templates can be prioritized for yield optimization.

7.3 Can PCR products be used directly in CFPS reactions?

Yes, but the promoter, translation initiation element, and termination structure must be complete, and inhibition caused by PCR residues must be controlled. Linear PCR templates are susceptible to nuclease degradation. If necessary, protective sequences should be added, template purity should be improved, or a system more suitable for linear templates should be selected.

7.4 Why are liposomes or nanodiscs needed when expressing membrane proteins by CFPS?

Membrane proteins contain hydrophobic transmembrane regions and are prone to aggregation or misfolding in aqueous systems. Liposomes, nanodiscs, detergents, or microsomal membranes can provide a membrane-mimicking environment, increasing the likelihood of membrane protein insertion, stabilization, and functional detection.

7.5 Why are disulfide bond-containing proteins often inactive in CFPS?

Disulfide bond formation requires a suitable redox environment and folding auxiliary factors. If the lysate is strongly reducing or lacks folding enzymes such as DsbC and PDI, the protein may be expressed but fail to adopt the correct conformation. Such proteins should be evaluated for both expression level and functional activity.

7.6 Why is CFPS suitable for unnatural amino acid incorporation?

CFPS is open and controllable, making it convenient to add unnatural amino acids, orthogonal tRNAs, and aminoacyl-tRNA synthetases, while also allowing adjustment of concentration and reaction conditions. Compared with cell-based systems, in vitro systems more readily avoid problems related to unnatural amino acid uptake, toxicity, and metabolic interference.

The value of cell-free protein synthesis systems lies in separating transcription, translation, and protein functional verification from the constraints of cell growth. For toxic proteins, membrane proteins, complex folded proteins, unnatural amino acid-containing proteins, and high-throughput screening tasks, CFPS can significantly shorten the design-expression-detection cycle.