A wheat germ cell-free expression system for SeMet labelling

Technology

Protein Expression

Summary

We have shown that the wheat germ cell-free protein production platform used by CESG can be used for efficient production of [Se-Met]-labeled proteins with incorporation efficiency very close to 100%.

Description

One of the major bottlenecks in high-throughput structure determination effort is protein expression. Structural proteomics efforts will require expression and purification of thousands of proteins and/or protein fragments. The ability to obtain labeled proteins is an essential aspect required for rapid progress in structural determinations. Critical examples are: (1) The production of U-15N labeled protein to assess the foldedness and aggregation state; (2) The production of U-13C;15N or selectively labeled proteins for NMR structure determinations; (3) The production of Se-Met labeled protein for phase determination in X-ray studies. The procedure from DNA to purified protein can be carried out in automated fashion on the CellFree Sciences Protemist DT-II(TM) bench-top robot in sufficient quantities for small-scale crystallization screening. However, 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.

Publication

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. PubMed ID: 16930128 | Search SBKB Publications portal

[1] Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 1(2):149-53 (2004). PubMed ID: 15782178 | Search SBKB Publications portal

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. PubMed ID: 15966852 | Search SBKB Publications portal

Contact

John L. Markley (markley@nmrfam.wisc.edu) or Brian G. Fox (bgfox@biochem.wisc.edu)

Center for Eukaryotic Structural Genomics

Availability

email for details

Link




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Last edited:Thu 27 Sep 2012 - 4 years, 5 months ago