Monitoring a dynamic system by liquid chromatography-mass spectrometry

Abstract

The present invention provides a method for monitoring of profile changes of components in a dynamic system such as a cell-free in vitro protein synthesis system by using liquid chromatography (LC) combined with mass spectrometry (MS). In an additional aspect, this invention provides a method for enhancing the yield and/or reproducibility in a cell-free protein synthesis system by modulating the level and/or activity of a protein component that has regulatory effects on the system.

1 .- 34 . (canceled) 
     
     
         35 . A modified prokaryotic cell lysate comprising a reduced amount of CspE protein as compared to a lysate prepared from a wild-type prokaryotic cell by the same method. 
     
     
         36 . The modified prokaryotic cell lysate of  claim 35  further comprising a reduced amount of CspA protein as compared to a lysate prepared from a wild-type prokaryotic cell by the same method. 
     
     
         37 . The modified prokaryotic cell lysate of  claim 35 , wherein the cell from which the lysate is prepared comprises a recombinant genomic modification that abolishes CspE expression. 
     
     
         38 . The modified prokaryotic cell lysate of  claim 36 , wherein the cell from which the lysate is prepared comprises a recombinant genomic modification that abolishes CspE expression and a recombinant genomic modification that abolishes CspA expression. 
     
     
         39 . The modified prokaryotic cell lysate of  claim 35  comprising iodoacetamide modified protein. 
     
     
         40 . The modified prokaryotic cell lysate of  claim 35  comprising T7 RNA polymerase. 
     
     
         41 . The modified prokaryotic cell lysate of  claim 35  comprising an exogenous nucleic acid template, wherein the exogenous nucleic acid template encodes a recombinant protein. 
     
     
         42 . The modified prokaryotic cell lysate of  claim 41  comprising the recombinant protein encoded by the exogenous nucleic acid template. 
     
     
         43 . A method of making a modified prokaryotic cell lysate, the method comprising:
 (i) providing a modified prokaryotic cell comprising a recombinant genomic modification that abolishes CspE expression; and   (ii) lysing the cell,   
       thereby forming the modified prokaryotic cell lysate. 
     
     
         44 . The method of  claim 43 , wherein the modified prokaryotic cell comprising a recombinant genomic modification that abolishes CspE expression further comprises a recombinant genomic modification that abolishes CspA expression. 
     
     
         45 . The method of  claim 43 , wherein the method further comprises activating the modified prokaryotic cell lysate by incubating the modified prokaryotic cell lysate at a temperature of at least 25° C. 
     
     
         46 . The method of  claim 43 , wherein the method further comprises contacting the modified prokaryotic cell lysate with iodoacetamide. 
     
     
         47 . The method of  claim 46 , wherein the method further comprises contacting the modified prokaryotic cell lysate with glutamate, pyruvate, AMP, GMP, UMP, CMP, and oxalate. 
     
     
         48 . The method of  claim 47 , wherein the method further comprises contacting the modified prokaryotic cell lysate with magnesium, ammonium, potassium, phosphate, and sodium. 
     
     
         49 . The method of  claim 48 , wherein the method further comprises contacting the modified prokaryotic cell lysate with putrescine and spermidine. 
     
     
         50 . The method of  claim 43 , wherein the method further comprises contacting the modified prokaryotic cell lysate with an exogenous nucleic acid template, wherein the exogenous nucleic acid template encodes a recombinant protein.