Design, Assemble and Express with Confidence: Enabling High-throughput DNA Assembly and Protein Expression Workflows

Protein expression can provide crucial insights for analyzing and understanding protein structure, protein function and gene regulation. Multiple methodologies exist that provide control over sequence, tags, signals and other characteristics, but typically use live cells. Unfortunately, live cell workflows limit throughput due to their multi-day cloning, transformation, expression and purification process. Additionally, live cells are limited by the toxicity of certain sequences.

To avoid these limitations, in vitro workflows utilizing cell-free protein synthesis (CFPS) can be employed to cut the assembly-to-expression timeframe down to one day. This enables the rapid screening of multiple protein designs and variants generated on demand, making it ideal for synthetic biology and protein engineering. 

In this blog, we showcase our cell-free workflow streamlined from design to purified protein. This drastically cuts time from the typical seven-day traditional cell-based workflow and is highly compatible with automation, enabling higher throughput and productivity.

 

From assembly design to protein purification

(1) Design your assemblies

NEB offers several open-access tools to simplify the design process for DNA assembly and to provide primer sequences for amplicon PCR. Utilizing design tools sets your experiments up for success.

 

NEBuilder^® Assembly Tool

The NEBuilder Assembly Tool uses your fragment sequences and the planned polymerase for amplification to quickly and easily design primers for NEBuilder HiFi DNA Assembly. You can then export your required oligos and final assembled sequence.

 

NEBridge^® Golden Gate Assembly Tool

The NEBridge Golden Gate Assembly Tool provides a visualization of your Golden Gate Assembly design and helps with primer design based on input sequences and Type IIS restriction enzyme. Additionally, this tool can be used to predict fusion site overhang fidelity or choose overhangs for the maximum expected ligation fidelity of the assembly.

 

NEBridge^® Ligase Fidelity Tools

The NEBridge Ligase Fidelity Tools suite helps you design complex, high-fidelity assemblies under different experimental conditions and check for potential fidelity or efficiency issues. These tools allow you to visualize overhang ligation preferences, predict high-fidelity junction sets, and split your sequence for scarless assembly.

 

(2) Obtain high-quality DNA

Once you have your assembly designed, there are a few options for obtaining your DNA. You can use PCR amplicons, restriction enzyme-digested DNA fragments or buy synthetic DNA from a synthesis provider. When carrying out PCR, we recommend using Q5^® Hot Start High-Fidelity 2X Master Mix (NEB #M0494). Although some assembly methods such as our NEBuilder HiFi DNA Assembly Master Mix allow you to skip the purification step under certain reaction volume conditions, we generally recommend using the Monarch^® Spin DNA Gel Extraction Kit (NEB #T1120) or Monarch Spin PCR & DNA Cleanup Kit (5 µg) (NEB #T1130) on your PCR amplicons to ensure integrity and purity.

 

(3) Assemble your DNA

There are several options for DNA assembly, such as NEBuilder HiFi DNA Assembly and NEBridge Golden Gate Assembly. These high-fidelity and high-efficiency assembly methods result in products that can be used directly in rolling circle amplification (RCA), which can then template cell-free protein expression.

NEBuilder HiFi DNA Assembly is used for seamless DNA assembly regardless of fragment length or end compatibility. This method is highly accurate, so it can be scaled to assemble constructs in parallel and is often used to prototype protein variants for engineering or screening. NEB offers the NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) for cell-free workflows. For more details on using NEBuilder HiFi DNA Assembly for protein expression, please refer to our application note on streamlining cell-free protein expression workflows with NEBuilder HiFi DNA Assembly.

 

Application note: A Streamlined Cell-Free Workflow for On-Demand Protein Expression Using NEBuilder^® HiFi DNA Assembly

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Golden Gate Assembly uses Type IIS restriction enzymes to cleave DNA for scarless, modular assembly. This method works efficiently for both low complexity (2 fragments) and high complexity (7-50+ fragments) assemblies while remaining robust and user-friendly. NEB offers two kits with our optimized NEBridge reagents: the NEBridge Golden Gate Assembly Kit (BsaI-HF^® v2) (NEB #E1601) and the NEBridge Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602). Alternatively, you can use your choice of NEB Type IIS enzyme with NEBridge Ligase Master Mix (NEB #M1100). For more details on using NEBridge Golden Gate Assembly in this workflow, please refer to our application note on our one-day workflow for DNA construction to protein expression.

 

Application note: Accelerating DNA Construction to Protein Expression – A Rapid 1-Day Workflow Using NEBridge^® Golden Gate Assembly

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(4) Rolling Circle Amplification (RCA)

Most cell-free protein synthesis (CFPS) systems require more DNA than is typically available directly from DNA assembly reactions, requiring amplification beforehand. RCA is a robust and highly sensitive isothermal amplification option for CFPS, as it continually and quickly amplifies circular DNA. This preferential amplification also enriches only successful ligation products, avoiding the need for additional cleanup prior to CFPS. The phi29-XT RCA Kit (NEB #E1603) includes an engineered polymerase for improved yield, reaction time, thermostability and sensitivity, achieving the desired yield in as little as two hours.

 

(5) Cell-free Protein Synthesis (CFPS)

To further cut down on time, the NEBExpress^® Cell-free E. coli Protein Synthesis System (NEB #E5360) expresses proteins ranging from 17-230 kDa in 2-4 hours by employing an extract-based, transcription/translation (TXTL) system. This CFPS method is also compatible with toxic or otherwise difficult proteins and allows for affinity purification methods. It can be miniaturized for high-throughput screening or scaled up for automated magnetic particle-based purification.

 

(6) Protein purification

Lastly, cell-free expressed polyhistidine-tagged, SNAP-tag^®, or maltose binding protein (MBP) fusion proteins can quickly and easily be purified using NEBExpress Ni-NTA Magnetic Beads (NEB #S1423), SNAP-Capture Magnetic Beads (NEB #S9145), or Amylose Magnetic Beads (NEB #E8035), respectively. Additionally, this workflow is compatible with both purifying soluble proteins and generating immobilized protein products.

 

Advancing throughput with workflow automation and miniaturization

In addition to accelerating the workflow by eliminating live-cell transformation and screening, each step is automation-compatible and can have higher throughput, especially when miniaturized.

Both Golden Gate Assembly and NEBuilder HiFi DNA Assembly enable the construction of complex DNA constructs and combinatorial libraries when scaled. These methods also support high-throughput plasmid construction and multi-site directed mutagenesis, streamlining protein variant design and testing.

CFPS can be performed with automated liquid handlers. Acoustic droplet ejection permits precise small volume transfer, so miniaturized volumes can increase throughput, reproducibility and accuracy. Additionally, resource availability is a common limitation in CFPS, so achieving 50-100 times more reactions per run makes high-throughput screening more cost-effective and scalable. Magnetic bead purification can be automated with magnetic particle processors or magnetic module decks to purify your protein after CFPS.

For more information about increasing throughput, please refer to our application note on automating cell-free protein expression and purification.

 

Application note: Automated Cell-free Protein Expression and Purification for High-Throughput Screening using NEBExpress^® Cell-free E. coli Protein Synthesis System and NEBExpress Ni-NTA Magnetic Beads

View Application Note