Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis
Methods and kits are provided for calibrating a cell-free protein synthesis reaction for optimal activity. The method includes the steps of providing an extract competent for cell-free protein synthesis (CFPS); performing cell-free protein synthesis with the extract; measuring a first reaction end-point where in vitro protein synthesis plateaus; measuring a second reaction end-point where Energy Charge of the extract declines to a level in a range from about 0.40 to about 0.80 of Energy Charge of a control extract; and adjusting Energy Charge of the extract to a level in a range from about 0.80 to about 1.0 of the control extract.
1. A method for calibrating a cell-free protein synthesis reaction for optimal activity, comprising:
(a) providing a yeast cell extract competent for cell-free protein synthesis (CFPS);
(b) performing a CFPS reaction with the extract;
(c) measuring a first reaction end-point where in vitro protein synthesis plateaus;
(d) measuring a second reaction end-point where Energy Charge of the extract declines to a level in a range from about 0.40 to about 0.80 of Energy Charge of a control extract, wherein the control extract is a separate extract not used in a CFPS reaction or the control extract is the extract provided for the CFPS reaction prior to performing the CFPS reaction;
(e) after the Energy Charge of the extract provided for the CFPS reaction declines to a level in a range from about 0.40 to about 0.8 of the Energy Charge of the control extract, adjusting Energy Charge of the extract provided for the CFPS reaction to a level in a range from about 0.80 to about 1.0 of the control extract, wherein adjusting comprises adding to the extract a phosphate-containing energy source selected from ATP, creatine phosphate, and a combination thereof; and
(f) removing a toxic metabolic byproduct from the CFPS reaction, wherein the toxic metabolic byproduct comprises inorganic phosphate.
2. The method of claim 1 , wherein the extract competent for cell-free protein synthesis and the control extract comprise extracts derived from a common extract source.
3. The method of claim 1 , wherein the yeast is Saccharomyces cerevisiae.
4. The method of claim 1 , wherein adjusting the Energy Charge of the extract comprises adjusting the Energy Charge before a time selected from the first reaction end-point and second reaction end-point.
5. The method of claim 1 , wherein adjusting the Energy Charge of the extract further comprises adding a phosphate-free energy source to the extract and the phosphate-free energy source is selected from the group consisting of glucose, starch and dextran.
6. The method of claim 1 , wherein adjusting the Energy Charge of the extract further comprises adding cAMP to the extract.
7. The method of claim 1 , wherein performing cell-free protein synthesis with the extract comprising performing a coupled transcription/translation reaction.
8. The method of claim 7 , wherein the coupled transcription/translation reaction comprises an expression template comprising SEQ ID NO:6 and a polymerase comprising phage T7 DNA polymerase.
9. The method of claim 1 , wherein the cell-free protein synthesis reaction is selected from a batch CFPS reaction or a semi-continuous CFPS reaction.