Cell-Free Protein Production

In cooperation with Cell-Free Science Technology Research Center at Ehime University (CFSTRC) (Matsuyama, Japan) and the Cell-Free Sciences (Yokohoma, Japan), we are investigating the potential of wheat germ cell-free protein translation as an enabling technology for structural genomics. Two important applications of this technology are a facile assessment of the ability of a given gene to be expressed and the ability to produce isotopically labeled proteins required for certain critical aspects of protein structure determination. A successful implementation of cell-free protein expression may minimize problems in cell harvesting, cell lysis, and pre-column manipulations. Moreover, this approach may simplify purification efforts as the protein of interest can become the dominant protein in the reaction mixture. Cell-free systems may also permit labeling strategies that cannot be achieved in whole cell systems, while potentially providing a substantial economy in the labeled material required to provide a target protein.

So far, studies carried out at the CESG with this technology on the full-length protein targets from Arabidopsis thaliana have yielded the following results:

Screening of 151 targets containing an N-terminal (His)6-tag on a 50-µL scale gave high-yield expression of highly soluble proteins with a success rate of 50%; screening of 109 targets containing an N-terminal GST-tag resulted in production of highly soluble protein following cleavage of GST fusion tag with PreScission™ Protease (Amersham Biosciences) with a success rate of 49%.

Fifty-two proteins containing an N-terminal (His)6-tag were expressed on a large scale (4-ml) using ¹⁵N-labeled amino acids; of these, 46% were obtained in quantities (0.6 mg/ml average yield of purified protein) sufficient for ¹H-¹⁵N HSQC NMR screening; with 58% of those proteins were folded, non-aggregating. Eight folded proteins have been expressed in mg quantities using ¹³C; ¹⁵N-labeled amino acids; from these, two three-dimensional structures have been solved, four others are in progress, and two proteins were obtained in yields insufficient for structure determination. Six proteins containing an N-terminal GST-tag were expressed on large scale (4-ml) using ¹⁵N-labeled amino acids; of these, 100% were obtained in quantities (0.5 mg/ml average yield of purified target protein) sufficient for ¹H-¹⁵N HSQC NMR screening; 33% of those proteins were folded, non-aggregating. One of these folded proteins has been expressed in mg quantities using ¹³C; ¹⁵N-labeled amino acids; one structure is in progress.

Advantages

• Wheat germ cell-free method supports rapid and efficient screening (supported by robotics).
• Wheat germ cell-free method requires smaller volumes. (avoids lengthy concentration steps in protein purification).
• Labeled proteins can be prepared rapidly (in 1-2 days) to meet needs of structural biologists.
• Supports labeling strategies that are not practical for proteins produced from bacterial cells (no label scrambling).
• Supports the production of eukaryotic N-terminal (His)6 proteins (previous experience showed that these were not produced successfully from E. coli cells).

Disadvantages

• Reagent intensive.
• Currently not compatible with Gateway cloning technology used in other parts of the CESG project.

CESG utilizes wheat germ cell-free instrumentation  from CellFree Sciences (Yokohoma, Japan). Pictured at right are the GeneDecoder1000 Fully Automated Protein Synthesizer (LEFT) and the Protemist Protein Synthesizer (RIGHT). Both systems make use of wheat germ translation technology developed by CESG collaborator Professor Yaeta Endo, Director of the Cell-Free Science Technology Research Center at Ehime University (Matsuyama, Japan). The GeneDecoder1000, which performs steps needed for transcription (DNA to mRNA) and translation (mRNA to protein) in 96-well format, enables up to 384 50-microliter reactions to be carried out overnight. CESG uses this instrument to screen genes or gene fragments to determine the level of protein production, whether a fusion product is cleavable, and the fraction of protein product that is soluble. The Protemist Protein Synthesizer automates up to eight 5-ml translation reactions used in producing proteins for structural investigations. In some cases, a single 5-ml reaction produces sufficient protein for an NMR structure determination. Proteins that express at lower levels require up to three 5-ml reactions.

CESG has carried out a detailed comparison of its protein production pipelines based on E. coli cells and on wheat germ cell-free translation. The E. coli cells portion of the experiment made use of CESG's standard maltose binding fusion containing a (His)6-tag and a TEV protease cleavage site. All targets found to be produced as a soluble, cleaved product were prepared with uniform [¹⁵N]-labeling. The cell-free part of the experiment compared two constructs for each of the 96 targets: one with a non-cleavable N-terminal (His)6-tag, and one with a cleavable (PreScission Protease, Amersham Biosciences) N-terminal GST-tag. These constructs were first screened on a small scale (50-mL) to determine the level of protein produced and its solubility. Targets that produced soluble protein were then produced as [U-¹⁵N]-proteins in larger scale (4 to 12 mL) cell-free translation reaction mixtures that contained ¹⁵N-labeled amino acids. The proteins produced by each method were analyzed by ¹H-¹⁵N correlation NMR spectroscopy for folding, aggregation state, and stability.

This project, which was completed in August, 2004, has provided a rich source of information. We are only beginning to mine the results to learn what they tell us about these different approaches.  When the success rates of individual steps, supplies, and labor are taken into account, the costs for making labeled proteins for NMR structure determinations by the two platforms (E. coli cells and wheat germ cell-free) are equivalent. The potential advantage of the cell-free approach is that nearly twice as many of the protein targets in this study prepared by cell-free than by E. coli cells yielded samples suitable for NMR structure determination. However, when the E. coli cell-free pipeline works, the yields of labeled proteins are higher.

In collaboration with Professor Yaeta Endo (Ehime University, Matsuyama, Japan) and CellFree Sciences (Yokohama, Japan), CESG has developed a platform that utilizes wheat germ cell-free technology to produce protein samples for NMR structure determinations. In the first stage, cloned DNA molecules coding for proteins of interest are transcribed and translated on a small scale (25 microL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2-10 microgram) is sufficient to be visualized by polyacrylamide gel electrophoresis. The fraction of soluble protein is estimated by comparing gel scans of total protein and soluble protein. Targets that pass this first screen by exhibiting high protein production and solubility move to the second stage. In the second stage, the DNA is transcribed on a larger scale, and labeled proteins are produced by incorporation of [¹⁵N]-labeled amino acids in a 4 mL translation reaction that typically produces 1-3 mg of protein. The [¹⁵N]-labeled proteins are screened by ¹H-¹⁵N HSQC NMR spectroscopyto determine whether the protein is a good candidate for solution structure determination. Targets that pass this second screen are then translated in a medium containing amino acids doubly labeled with ¹⁵N and ¹³C. These steps can be automated so that the labor costs involved are minimal. CESG uses an automated platform for wheat germ cell-free production of labeled proteins. Our current robotic systems from CellFree Sciences (Yokohoma, Japan) include the GeneDecoder1000 (2-5 ug per well in 96-well format), the Protemist10 and the Protemist100 (1-2 mg per sample in eight samples format), and Protemist DT-II (0.1-0.3 mg purified protein per well in 6-well format).

The GeneDecoder1000 is used to produce samples to screen for expression, solubility and, where appropriate, tag cleavage. The Protemist10 and the Protemist100, coupled with ACTA PRIME purification systems, are used for expression and purification of sufficient quantities of labeled protein for NMR structural studies. Our cumulative experience with cell-free expression includes over 1000 different structural genomics targets from human, mouse, Plasmodium, and Arabidopsis. To date, CESG has deposited into the PDB 23 NMR structures of eukaryotic proteins produced by wheat germ cell-free methodology. The average yield of labeled purified proteins has been ~1.2 mg per ml of wheat germ lysate (OD260=200). We also report that the Protemist DT-II provides a cost-effective and rapid method for screening multiple constructs engineered to improve solubility or foldedness.

Furthermore, CESG has begun structural investigations of membrane proteins using the automated translation and purification capabilities of the Protemist DT-II. Several detergents have been identified to be compatible with wheat germ cell-free translation, and current efforts are aimed at developing efficient ways of protein concentration, detergent exchange, and preparation of structural samples (unpublished results).

Vinarov, D.A., Loushin Newman, C.L., Tyler, E.M., Markley, J.L. (2006) Wheat germ cell-free expression system for protein production. Current Protocols in Protein Science, Wiley Interscience.

Vinarov, D.A., Lytle, B.L., Peterson, F.C., Tyler, E.M., Volkman, B.F., Markley, J.L. (2004) Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 1(2):149-53. |15782178|

Vinarov, D.A., Markley, J.L. (2005) High-throughput automated platform for nuclear magnetic resonance-based structural proteomics. Expert Rev Proteomics 2(1):49-55. |15966852|

Vinarov, D.A., Newman, C.L., Markley, J.L. (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J 273(18):4160-9. |16930128|