Cube E.coli T7 lysates for cell-free protein expression

 

For increased convenience and higher protein yields (up to 6 mg/ml GFP),  the HiYield-T7 lysate was developed. It already includes the T7 RNA polymerase enzyme.

 

As an alternative to protein expression in living cells, cell-free lysates can be used to obtain recombinant proteins. They are particularly useful for the following applications:

 

  fast screening of multiple expression constructs

  introduction of unnatural amino acids, labels (biotin, fluorescent markers)

  labelling of proteins for NMR

  direct integration of membrane proteins into nanodiscs

 

Cell-free protein expression

 

For expression reactions that do not use the T7 RNA promoter, please refer to the standard and HiYield E.coli lysates.

 

In close collaboration with the group of Volker Doetsch and Frank Bernhard, Institute of Biophysics at the University of Frankfurt, Germany, we provide an open system that can be adapted for a range of applications, with dedicated protocols for membrane proteins, continous exchange (dialysis) reactions, and many more.  This system takes more work initially than ready to use kits, but provides ultimate flexiblity in reaction variations.

 

Please note that the E.coli T7 lysates require additional components (e.g. amino acids, nucleic acids, dialysis devices) that are currently not provided by Cube Biotech. Please refer to the protocols for more details.

 

  Quality control of cell-free expression lysates
Fig. 1: Cell-free expression of GFP with HiYield-T7  lysates. Variation of Mg2+ concentration (14, 16, 18, 20,22 mM). A Mg2+ optimum was observed at ca. 18 mM for combinations of cell-free lysate and other reaction components. Yields of up to 6 mg/ml could be obtained with the HiYield-T7 lysate. Data kindly provided by Frank Bernhard, University of Frankfurt, Department of Biophysical Chemistry.
 
  Successful protein crystallization after cell-free expression
Fig. 2:
X-ray structure of cell-free-expressed diacylglycerol kinaseDgkA to 2.28 Å resolution. The protein was P-CF expressed in HiYield-T7 lysate. The integral membrane enzyme is shown as a ribbon model viewed from the membrane plane. The individual subunits of the DgkA trimer are coloured blue, green and orange. Lipid molecules are shown as stick models with carbons and oxygens colored yellow and red, respectively. The N- and C-termini of one subunit are indicated (Boland et al., (2014) CMLS ).
Data kindly provided by Frank Bernhard, University of Frankfurt, Department of Biophysical Chemistry.
 
       

 

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E.coli cell lysates-T7

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