University of Wisconsin–Madison
Protein production

NMRFAM Protein Production Methods


In order to produce sufficient mass of bacterial cells to facilitate subsequent protein purification efforts for functional studies and structure determination, cell-based or cell-free translation methods are currently being used to prepare proteins. However, with conventional cell-based methods, many individual proteins cannot be expressed in soluble form in bacteria and are, therefore, not suitable for E. coli cell-based production methodologies. Insolubility arises either from an intrinsic property of a protein (for example, aggregation due to a very hydrophobic patch on the surface) or because the protein is not susceptible to the folding mechanisms in the expression host; in which case there is an aggregation of folding intermediates. These include one-third to one half of prokaryote proteins. This proportion is likely to be higher for eukaryotic proteins, particularly those that comprise multiple domains, those that require cofactors or protein partners for proper folding, or those that require extensive post-translational modification. The development of new systems and strategies capable of synthesizing any desired soluble, labeled protein, or protein fragment on a preparative scale as alternatives E. coli cell-based production is one of the most important tasks in biotechnology today.


We are developing cell-free translation, which utilizes ENDEXT® technology, as a versatile method for studying protein structure and function. This experimental platform is based on establishing predictive behaviors for membrane proteins using robotic cell-free translation in small-scale (50 µL to 1.2 mL). Robotic translation has an advantage of simplicity and reproducibility in the provision of material used for further research and protocol development. When appropriate expression and solubilization conditions are identified, purification protocols are developed in small-scale, and then the work is transferred to different robots whose operational volume (1.2 mL to 20 mL) better supports structure determination or expanded biological studies.

Robotic Instrumentation

The unique suite of cell-free translation robots were developed and manufactured by the CellFree Sciences Co., Ltd. (Yokohama, Japan). They are used for high-throughput screening (GenDecoder1000) or scale-up production (Protemist DTII) of milligram quantities of membrane protein for structural analysis. The newest robot, the Protemist XE, operates using a tangential flow dialysis platform and can synthesize up to 50 mg of membrane proteins in 48 h. These instruments, and the personnel and expertise to operate them, were assembled during PSI-1 and PSI-2 at the Center for Eukaryotic Structural Genomics (CESG). No other freely accessible research site exists worldwide with this capability.


gendecoder 1000GenDecoder1000

Typical use: HTP screening

Format: 96 well; 4×96 well

Total reaction volume: 14.4-19.2 mL

Reaction time: 24 h

Automated transcription: yes

Automated translation: yes

Automated purification: no

Yield per reaction: 1 to 5 µg

protemistdtIIProtemist DTII

Typical use: screening; characterization

Format: 24 well; 6 well

Total reaction volume: 33.6 mL; 24 mL

Reaction time: 14 to 24 h

Automated transcription: yes

Automated translation: yes

Automated purification: no

Yield per reaction: 50 µg to 1 mg

Protemist 100Protemist100

Typical use: characterization; production

Format: 8 tubes

Total reaction volume: 32 mL

Reaction time: 18 h

Automated transcription: yes

Automated translation: yes

Automated purification: no

Yield per reaction: 0.5 to 10 mg

protemistxeProtemist XE

Typical use: production

Format: 1 reaction

Total reaction volume: 10-20 mL

Reaction time: 12 to 48 h

Automated transcription: no

Automated translation: yes

Automated purification: no

Yield per reaction: 1 to 70 mg

Cell-Free Publications

Beebe ET, Makino SI, Markley JL, Fox BG (2014) Automated cell-free protein production methods for structural studiesPharmacol Toxicol 1140:117-35.

Beebe ET, Makino S-I, Nozawa A, Matsubara Y, Frederick RO, Primm JG, Goren MA, Fox BG (2010) Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteinsN Biotechnol 28(3):239-49.

Makino SI, Beebe ET, Markley JL, Fox BG (2014) Cell-free protein synthesis for functional and structural studiesMethods Mol Biol 1091:161-78.

Makino S-i, Goren MA, Fox BG, Markley JL (2010) Cell-free protein synthesis technology in NMR high-throughput structure determinationMethods Mol Biol 607:127-47.