Cell-free protein expression is a great complement to in vivo expression systems such as bacteria or insect cell for the production of selectively labeled recombinant proteins. Indeed, cell-free expression does not require cell culture nor extensive protein purification thus enables cost effective and straightforward labeling with the incorporation of any type of labeled or unnatural amino acids.
Musaibo Kun cell-free kits have been designed for coupled in vitro transcription and translation of circular or linear DNA into proteins in a single reaction system. The kits exploit an optimized E. coli cell extract and typically contain an ATP-regeneration system along with reference DNA, dialysis devices and a mixture of unlabeled amino acids. In addition, cell-free kits are supplemented with scrambling inhibitors to avoid isotope dilution.
Together with collaborators at Taiyo Nippon Sanso, CEA and Pasteur Institute (France), Cortec Isotope has developed a robust protocol for cell-free expression of stable isotope labeled proteins using Musaibo Kun CFPS kits. Special emphasis was placed on increasing expression yields employing AT-rich codons and optimized vectors.
The developed protocol typically follows a two-step strategy, which first consists in optimizing protein expression conditions using a small-scale system (starter kit). Starter kits have been employed to determine ideal parameters for optimal expression of protein kinase p38a.
SDS page of p38a (MW = 41 kDa) expressed with a cell-free starter kit.
Incorporation of stable isotopes into protein samples is performed with large scale cell-free kits that allow the production of a few mg of protein. Cell-free expression was used for straightforward and cost-effective deuteration of 15N labeled p38a, leading to substantial improvement of the quality of 1H/15N TROSY NMR spectrum along with reduction of experimental time.
1H/15N TROSY NMR spectra of p38a recorded at 700 MHz. Left: 15N,1H p38a expressed in E. coli (Exp time: 10 h, sample conc. 100 uM, sample vol. 300 uL). Right: 15N,2H p38a expressed with cell-free kit (Exp time: 2h30, sample conc. 50 uM, sample vol. 100 uL).
Stable isotope enriched amino acids must be purchased separately to perform labeled protein expression with cell-free kits. Cell-free expression is particularly well-adapted to the use of the SAIL strategy for detailed structure determination of proteins up to 50 KDa. Cost-effective specific deuteration can be performed using 15N, Da amino acids, allowing for a slower T2 relaxation leading to an increase of NMR signal intensity up to 60%.
Cortec Isotope is pleased to offer cell free kits along with labeled amino acids, SAIL amino acids as well as the 20xAA mixture.
Cortec Isotope proposes to help you label your protein. For inquiry, please contact our labeled protein expression platform.
