Functional protein expression from a DNA based wheat germ cell-free system.
Wheat germ based eukaryotic cell-free systems have been shown to be applicable for both functional and structural analyses of proteins. However, the existing methods might require specialized instrumentation and/or a separate mRNA synthesis step. We have developed a DNA based, highly productive, coupled transcription/translation wheat germ cell-free system that incorporates the normally separate mRNA synthesis step and does not require specialized instrumentation. Using a small-volume batch reaction with fluorescence labeling, DNA templates predicted to encode proteins could be quickly screened for their ability to direct the expression of proteins of the appropriate size. Protein yield can be increased as much as 2 to 4-fold in this system using a dialysis reaction, reaching approximately 200-440 microg/ml in 10-20 h. Furthermore, enzyme activities can be assayed directly in the extract without further purification. Simple purification with affinity tags can be achieved in one-step and with minor modifications, efficient SeMet and [U-15N] labeling of >95% can be accomplished in this system. Thus, this efficient cell-free expression system can facilitate both functional and structural proteomics.
Zhao
Kate Qin
KQ
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA. kate.zhao@promega.com
Hurst
Robin
R
Slater
Michael R
MR
Bulleit
Robert F
RF
D053148:Caspase 3 / Q000378:metabolism; D002474:Cell-Free System; D018744:DNA, Plant / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D018745:Genome, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000378:metabolism*
D018744:DNA, Plant; D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D053148:Caspase 3