From idea to protein faster. How to get the most out of your workflow.
Classical cell-based protein expression is time-consuming. Cloning, transformation, expression, and purification often take several days. Furthermore, some constructs can be toxic in cells, hindering your workflow.
Cell-free protein synthesis (CFPS) makes this much faster. By first assembling DNA and then using it directly in a cell-free system, you can produce proteins the same day. This enables rapid variant screening and is ideal for synthetic biology and protein engineering.
In this blog post, we demonstrate how to streamline the cell-free workflow from design to purified protein. This approach shortens the traditional cell-based workflow from seven days to one and is ideally suited for automation, increased throughput, and increased productivity.
1. Design your constructs smartly
New England Biolabs (NEB) offers free tools that simplify the design process. These include:
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NEBuilder Assembly Tool for HiFi assembly
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NEBridge Golden Gate Assembly Tool for Type IIS-based designs
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NEBridge Ligase Fidelity Tools for complex, modular projects
These tools aid in primer design and increase your chances of successful assembly.
2. Create and purify your DNA
For best results, use PCR amplicons or sheared DNA fragments and purify your DNA, for example, with Monarch PCR & DNA Cleanup kits from NEB. High-quality input is essential for stable assembly and expression.
3. Assemble your DNA
Depending on your project, you choose:
- NEBuilder HiFi DNA Assembly Master Mix for seamless assembly and parallel variations
- NEBridge Golden Gate Assembly Kit for fast, modular assembly of 2 to more than 50 fragments
These high-fidelity and high-efficiency assembly methods yield products that can be used directly in Rolling Circle Amplification (RCA), after which they serve as a template for Cell-free Protein Synthesis (CFPS).
NEBuilder HiFi DNA Assembly is suitable for seamless DNA assembly regardless of fragment length or end compatibility. The method is highly accurate, allowing parallel assemblies to be easily scaled up. Ideal for prototyping protein variants for engineering and screening.
4. Multiply your DNA with RCA
CFPS often requires more DNA. Rolling Circle Amplification allows you to generate large quantities of circular DNA within 2 hours. Only successfully ligated constructs are amplified.
5. Cell-free Protein Synthesis (CFPS)
The NEBExpress Cell-free E. coli Protein Synthesis System allows you to synthesize proteins, including difficult or toxic proteins, in 2 to 4 hours. The system is scalable and suitable for automation.
6. Purify your protein
Use magnetic beads from NEBExpress Ni NTA, SNAP Capture, or amylose beads to rapidly isolate His-tag, SNAP-tag, or MBP fusions. This works for both low-volume and high-throughput applications.
Why this workflow works
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from days to hours
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ideal for variant screening
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compatible with automation
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cost reduction in miniaturization
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also works for challenging proteins
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